Nutritional compositions containing an elevated level of inositol and uses thereof

ABSTRACT

A method for enhancing neurological health and development in a subject involving administering to the subject a nutritional composition including no greater than about 7 g/100 kcal of a fat or lipid source; no greater than about 7 g/100 kcal of a protein or protein equivalent source; at least about 5 g/100 kcal of a carbohydrate; and at least about 9 mg/100 kcal of inositol, wherein the ratio of exogenous inositol to inherent inositol is at least 50:50.

TECHNICAL FIELD

The present disclosure relates to nutritional compositions that includeinositol at levels found to provide neurological benefits. Moreparticularly, the disclosed nutritional compositions include a fat, acarbohydrate, a protein or protein equivalent source, and inositol, forimproving neurological health and/or preventing or protecting againstthe development of neurodegenerative diseases. The nutritionalcompositions described herein are suitable for administration to adultand pediatric subjects.

BACKGROUND

The nervous system is responsible for accumulating and analyzing sensoryinput and coordinating the generation of the appropriate functionalresponse. The successful execution and integration of these activitiesis dependent on the transmission of neuronal action potentials,electrical signals required to generate functional outputs. While it isthe neuronal cell that is responsible for the actual conduction of thesignaling current, the rate at which the signal travels is greatlyenhanced by the insulating properties of its glial-derived myelinsheath. In the central nervous system (CNS), glial cells known asoligodendrocytes are responsible for the formation of myelin. Theseterminally differentiated cells arise from progenitors termedoligodendrocyte precursor cells (OPCs). During development, OPCs areexposed to proliferative signals as they migrate along axons throughoutthe CNS. These developmental cues help ensure that the extent of OPCproliferation is sufficient to generate the appropriate number ofoligodendrocytes to myelinate all relevant axonal segments. Once therequired number of precursor cells has been generated, thedifferentiation process initiates followed by myelination.

Therefore, the impacts on the myelination process by brain nutrients areintegrated by three basic aspects: (1) the survival and proliferation ofoligodendritic projector cells (OPCs); (2) the differentiation of OPCsinto oligodendric cells (Oligo); and myelination deposition.

Myelination and synapse development are very important for neurologicalhealth. This is true in infants and children, for brain development andto provide improvement in specific brain function such as cognition,memory function, learning capacity, social interaction skills, reducedanxiety, visual acuity, motor skills and/or language skills. Likewise,improved myelination and synapse development can be beneficial inadults, especially adults with neurodegenerative diseases likeAlzheimer's disease.

Myelination can be described as the process by which a fatty layer,called myelin, accumulates around nerve cells (neurons), and begins ininfancy and continues through adulthood. Myelin particularly formsaround the long shaft, or axon, of neurons. Myelination enables nervecells to transmit information faster and allows for more complex brainprocesses. Thus, the process is vitally important to healthy centralnervous system functioning.

In the nervous system, a synapse is a structure that permits a neuron topass an electrical or chemical signal to another cell (neural orotherwise). Thus, synapses are essential to neuronal function: neuronsare cells that are specialized to pass signals to individual targetcells, and synapses are the means by which they do so. At a synapse, theplasma membrane of the signal-passing neuron (the presynaptic neuron)comes into close apposition with the membrane of the target(postsynaptic) cell. Both the presynaptic and postsynaptic sites containextensive arrays of molecular machinery that link the two membranestogether and carry out the signaling process. Synapse development, orsynaptogenesis, is the formation of synapses between neurons in thenervous system. Although it occurs throughout a healthy person'slifespan, an explosion of synapse formation occurs during early braindevelopment, known as exuberant synaptogenesis.

Myelination begins in the brain stem and cerebellum before birth, but isnot completed in the frontal cortex until late in adolescence. Breastfeeding contributes to more rapid myelination in the brain.Identification of nutrients that promote survival and proliferation ofoligodendrocytes represents a major unmet need. Beyond normaldevelopment, white matter injury is one of the leading causes ofneurological disease in infants, especially in infants born premature.In these cases, oligodendrocyte cell death and a lack of developmentalmyelination represent leading factors, resulting in aberrant neuralcircuit formation and nervous system refinement.

Thus, it would be useful to provide nutritional compositions that areable to improve myelination and synapse development in a subject. Inparticular, it may be useful to provide improved neurological health andfunction, including cognition, language development and motor skills inearly life in order to reduce or prevent adult neurological diseases. Itwould also be useful to combat neurodegenerative disease throughimproved myelination and synapse development in adults.

Accordingly, the present disclosure provides a nutritional compositionincluding inositol as described herein. In some embodiments, thenutritional composition also includes a fat or lipid, carbohydrate andprotein or protein equivalent source.

BRIEF SUMMARY

Briefly, the present disclosure is directed, in an embodiment, to anutritional composition comprising exogenous inositol, and to a methodfor providing improved neurological health and function, includingcognition, language development and motor skills in early life in orderto reduce or prevent adult neurological diseases, in a pediatricsubject, the method comprising administering to the pediatric subject anutritional composition comprising inositol, a fat or lipid, a proteinor protein equivalent source and a carbohydrate. In some embodiments,the nutritional composition can also include one or more long chainpolyunsaturated fatty acids (LCPUFAs) selected from the group consistingof docosahexaenoic acid (DHA) and arachidonic acid (ARA),phosphatidylethanolamines (PE), sphingomyelin (SPM), alpha-lipoic acid(ALA), epigallocatechin gallate (EGCG), sulforaphane, or combinationsthereof, which may synergistically combine with inositol to furtherimprove neurological health and development.

In certain embodiments, the nutritional composition further comprises anenriched lipid fraction derived from milk. In some embodiments thenutritional composition may include an enriched lipid fraction derivedfrom milk that includes milk fat globules. The addition of the milk fatglobules provides an enriched fat and lipid source to the infant thatmay be more fully digested by a pediatric subject.

In embodiments, the enriched lipid fraction and/or the milk fat globulesmay include saturated fatty acids, trans-fatty acids, monounsaturatedfatty acids, polyunsaturated fatty acids, cholesterol, odd-branchedchain fatty acids “OBCFAs”, branched chain fatty acids “BCFAs”,conjugated linoleic acid “CLA”, phospholipids, or milk fat globulemembrane protein, and mixtures thereof.

It is to be understood that both the foregoing general description andthe following detailed description present embodiments of the disclosureand are intended to provide an overview or framework for understandingthe nature and character of the disclosure as it is claimed. Thedescription serves to explain the principles and operations of theclaimed subject matter. Other and further features and advantages of thepresent disclosure will be readily apparent to those skilled in the artupon a reading of the following disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a dose-response for inositol on purifiedoligodendrogial cultures, resulting from the testing of Example 1.

FIG. 2 illustrates the OPCs, normalized to 40 μM resulting from thetesting of Example 2.

FIG. 3 illustrates the Oligo Number, normalized to 40 μM resulting fromthe testing of Example 3.

FIG. 4 shows the dual-fluorescence labeling of OPCs and oligodendrytes,as well as myelin deposition, resulting from the testing of Example 4.

FIG. 5 illustrates a dose-response of inositol on myelin extent.

FIGS. 6 and 7 illustrate the Density Bassoon and Density Homer countsresulting from the testing of Example 6.

FIG. 8 illustrates the colocalization of synaptic sites resulting fromthe testing of Example 6.

FIG. 9 illustrates the synaptic site sizes resulting from the testing ofExample 6.

FIG. 10 shows the effects of inositol on synaptic development under theflorescent microscopy resulting from the testing of Example 6.

DETAILED DESCRIPTION

Reference now will be made in detail to the embodiments of the presentdisclosure, one or more examples of which are set forth hereinbelow.Each example is provided by way of explanation of the nutritionalcomposition of the present disclosure and is not a limitation. In fact,it will be apparent to those skilled in the art that variousmodifications and variations can be made to the teachings of the presentdisclosure without departing from the scope of the disclosure. Forinstance, features illustrated or described as part of one embodiment,can be used with another embodiment to yield a still further embodiment.

Thus, it is intended that the present disclosure covers suchmodifications and variations as come within the scope of the appendedclaims and their equivalents. Other objects, features and aspects of thepresent disclosure are disclosed in or are obvious from the followingdetailed description. It is to be understood by one of ordinary skill inthe art that the present discussion is a description of exemplaryembodiments only and is not intended as limiting the broader aspects ofthe present disclosure.

The present disclosure relates generally to nutritional compositionsthat are suitable for administration to a pediatric subject.Additionally, the disclosure relates to methods for improvingneurological health and development in children and adults viaadministration of the nutritional composition(s) disclosed herein.

“Nutritional composition” means a substance or formulation thatsatisfies at least a portion of a subject's nutrient requirements. Theterms “nutritional(s)”, “nutritional formula(s)”, “enteralnutritional(s)”, and “nutritional supplement(s)” are used asnon-limiting examples of nutritional composition(s) throughout thepresent disclosure. Moreover, “nutritional composition(s)” may refer toliquids, powders, gels, pastes, solids, concentrates, suspensions, orready-to-use forms of enteral formulas, oral formulas, formulas forinfants, formulas for pediatric subjects, formulas for children,growing-up milks and/or formulas for adults.

The term “enteral” means deliverable through or within thegastrointestinal, or digestive, tract. “Enteral administration” includesoral feeding, intragastric feeding, transpyloric administration, or anyother administration into the digestive tract. “Administration” isbroader than “enteral administration” and includes parenteraladministration or any other route of administration by which a substanceis taken into a subject's body.

“Pediatric subject” means a human no greater than 13 years of age. Insome embodiments, a pediatric subject refers to a human subject that isbetween birth and 8 years old. In other embodiments, a pediatric subjectrefers to a human subject between 1 and 6 years of age. In still furtherembodiments, a pediatric subject refers to a human subject between 6 and12 years of age. The term “pediatric subject” may refer to infants(preterm or full term) and/or children, as described below.

“Infant” means a human subject ranging in age from birth to not morethan one year and includes infants from 0 to 12 months corrected age.The phrase “corrected age” means an infant's chronological age minus theamount of time that the infant was born premature. Therefore, thecorrected age is the age of the infant if it had been carried to fullterm. The term infant includes low birth weight infants, very low birthweight infants, extremely low birth weight infants and preterm infants.“Preterm” means an infant born before the end of the 37th week ofgestation. “Late preterm” means an infant from between the 34th week andthe 36th week of gestation. “Full term” means an infant born after theend of the 37th week of gestation. “Low birth weight infant” means aninfant born weighing less than 2500 grams (approximately 5 lbs, 8ounces). “Very low birth weight infant” means an infant born weighingless than 1500 grams (approximately 3 lbs, 4 ounces). “Extremely lowbirth weight infant” means an infant born weighing less than 1000 grams(approximately 2 lbs, 3 ounces).

“Child” means a subject ranging in age from 12 months to 13 years. Insome embodiments, a child is a subject between the ages of 1 and 12years old. In other embodiments, the terms “children” or “child” referto subjects that are between one and about six years old, or betweenabout seven and about 12 years old. In other embodiments, the terms“children” or “child” refer to any range of ages between 12 months andabout 13 years.

“Children's nutritional product” refers to a composition that satisfiesat least a portion of the nutrient requirements of a child. A growing-upmilk is an example of a children's nutritional product.

The term “degree of hydrolysis” refers to the extent to which peptidebonds are broken by a hydrolysis method. The degree of proteinhydrolysis for purposes of characterizing the hydrolyzed proteincomponent of the nutritional composition is easily determined by one ofordinary skill in the formulation arts by quantifying the amino nitrogento total nitrogen ratio (AN/TN) of the protein component of the selectedformulation. The amino nitrogen component is quantified by USP titrationmethods for determining amino nitrogen content, while the total nitrogencomponent is determined by the Tecator Kjeldahl method, all of which arewell known methods to one of ordinary skill in the analytical chemistryart.

The term “partially hydrolyzed” means having a degree of hydrolysiswhich is greater than 0% but less than about 50%.

The term “extensively hydrolyzed” means having a degree of hydrolysiswhich is greater than or equal to about 50%.

The term “protein-free” means containing no measurable amount ofprotein, as measured by standard protein detection methods such assodium dodecyl (lauryl) sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) or size exclusion chromatography. In some embodiments, thenutritional composition is substantially free of protein, wherein“substantially free” is defined hereinbelow.

“Infant formula” means a composition that satisfies at least a portionof the nutrient requirements of an infant. In the United States, thecontent of an infant formula is dictated by the federal regulations setforth at 21 C.F.R. Sections 100, 106, and 107. These regulations definemacronutrient, vitamin, mineral, and other ingredient levels in aneffort to simulate the nutritional and other properties of human breastmilk.

The term “growing-up milk” refers to a broad category of nutritionalcompositions intended to be used as a part of a diverse diet in order tosupport the normal growth and development of a child between the ages ofabout 1 and about 6 years of age.

“Milk-based” means comprising at least one component that has been drawnor extracted from the mammary gland of a mammal. In some embodiments, amilk-based nutritional composition comprises components of milk that arederived from domesticated ungulates, ruminants or other mammals or anycombination thereof. Moreover, in some embodiments, milk-based meanscomprising bovine casein, whey, lactose, or any combination thereof.Further, “milk-based nutritional composition” may refer to anycomposition comprising any milk-derived or milk-based product known inthe art.

“Milk” means a component that has been drawn or extracted from themammary gland of a mammal. In some embodiments, the nutritionalcomposition comprises components of milk that are derived fromdomesticated ungulates, ruminants or other mammals or any combinationthereof.

“Fractionation procedure” includes any process in which a certainquantity of a mixture is divided up into a number of smaller quantitiesknown as fractions. The fractions may be different in composition fromboth the mixture and other fractions. Examples of fractionationprocedures include but are not limited to, melt fractionation, solventfractionation, supercritical fluid fractionation and/or combinationsthereof.

“Fat globule” refers to a small mass of fat surrounded by phospholipidsand other membrane and/or serum proteins, where the fat itself can be acombination of any vegetable or animal fat.

“Nutritionally complete” means a composition that may be used as thesole source of nutrition, which would supply essentially all of therequired daily amounts of vitamins, minerals, and/or trace elements incombination with proteins, carbohydrates, and lipids. Indeed,“nutritionally complete” describes a nutritional composition thatprovides adequate amounts of carbohydrates, lipids, essential fattyacids, proteins, essential amino acids, conditionally essential aminoacids, vitamins, minerals and energy required to support normal growthand development of a subject.

Therefore, a nutritional composition that is “nutritionally complete”for a preterm infant will, by definition, provide qualitatively andquantitatively adequate amounts of carbohydrates, lipids, essentialfatty acids, proteins, essential amino acids, conditionally essentialamino acids, vitamins, minerals, and energy required for growth of thepreterm infant.

A nutritional composition that is “nutritionally complete” for a fullterm infant will, by definition, provide qualitatively andquantitatively adequate amounts of all carbohydrates, lipids, essentialfatty acids, proteins, essential amino acids, conditionally essentialamino acids, vitamins, minerals, and energy required for growth of thefull term infant.

A nutritional composition that is “nutritionally complete” for a childwill, by definition, provide qualitatively and quantitatively adequateamounts of all carbohydrates, lipids, essential fatty acids, proteins,essential amino acids, conditionally essential amino acids, vitamins,minerals, and energy required for growth of a child.

As applied to nutrients, the term “essential” refers to any nutrientthat cannot be synthesized by the body in amounts sufficient for normalgrowth and to maintain health and that, therefore, must be supplied bythe diet. The term “conditionally essential” as applied to nutrientsmeans that the nutrient must be supplied by the diet under conditionswhen adequate amounts of the precursor compound is unavailable to thebody for endogenous synthesis to occur.

“Probiotic” means a microorganism with low or no pathogenicity thatexerts a beneficial effect on the health of the host.

The term “inactivated probiotic” means a probiotic wherein the metabolicactivity or reproductive ability of the referenced probiotic has beenreduced or destroyed. The “inactivated probiotic” does, however, stillretain, at the cellular level, its cell structure or other structureassociated with the cell, for example exopolysaccharide and at least aportion its biological glycol-protein and DNA/RNA structure. As usedherein, the term “inactivated” is synonymous with “non-viable”.

“Prebiotic” means a non-digestible food ingredient that beneficiallyaffects the host by selectively stimulating the growth and/or activityof one or a limited number of bacteria in the digestive tract that canimprove the health of the host.

“Inherent inositol”, “endogenous inositol” or “inositol from endogenoussources” each refer to inositol present in the composition that is notadded as such, but is present in other components or ingredients of thecomposition; the inositol is naturally present in such other components.Contrariwise, “exogenous” inositol is inositol which is intentionallyincluded in the nutritional composition of the present disclosureitself, rather than as an element of another component.

“Branched Chain Fatty Acid” (“BCFA”) means a fatty acid containing acarbon constituent branched off the carbon chain. Typically the branchis an alkyl branch, especially a methyl group, but ethyl and propylbranches are also known. The addition of the methyl branch lowers themelting point compared with the equivalent straight chain fatty acid.This includes branched chain fatty acids with an even number of carbonatoms in the carbon chain. Examples of these can be isomers oftetradecanoic acid, hexadecanoic acid.

“Odd- and Branched-Chain Fatty Acid” (“OBCFA”) is a subset of BCFA thathas an odd number of carbon atoms and have one or more alkyl branches onthe carbon chain. The main odd- and branched-chain fatty acids found inbovine milk include, but are not limited to, the isomers oftetradecanoic acid, pentadecanoic acid, hexadecanoic acid, andheptadecanoic acid. For the purposes of this disclosure, the term “BCFA”includes both branched-chain fatty acids and odd-and-branched chainfatty acids.

“Trans-fatty acid” means an unsaturated fat with a trans-isomer.Trans-fats may be monounsaturated or polyunsaturated. Trans refers tothe arrangement of the two hydrogen atoms bonded to the carbon atomsinvolved in a double bond. In the trans arrangement, the hydrogens areon opposite sides of the bond. Thus a trans-fatty acid is a lipidmolecule that contains one or more double bonds in trans geometricconfiguration.

“Phospholipids” means an organic molecule that contains a diglyceride, aphosphate group and a simple organic molecule. Examples of phospholipidsinclude but are not limited to, phosphatidic acid,phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine,phsphatidylinositol, phosphatidylinositol phosphate,phosphatidylinositol biphosphate and phosphatidylinositol triphosphate,ceramide phosphorylcholine, ceramide phosphorylethanolamine and ceramidephosphorylglycerol. This definition further includes sphigolipids,glycolipids, and gangliosides.

“Phytonutrient” means a chemical compound that occurs naturally inplants. Phytonutrients may be included in any plant-derived substance orextract. The term “phytonutrient(s)” encompasses several broadcategories of compounds produced by plants, such as, for example,polyphenolic compounds, anthocyanins, proanthocyanidins, andflavan-3-ols (i.e. catechins, epicatechins), and may be derived from,for example, fruit, seed or tea extracts. Further, the termphytonutrient includes all carotenoids, phytosterols, thiols, and otherplant-derived compounds. Moreover, as a skilled artisan will understand,plant extracts may include phytonutrients, such as polyphenols, inaddition to protein, fiber or other plant-derived components. Thus, forexample, apple or grape seed extract(s) may include beneficialphytonutrient components, such as polyphenols, in addition to otherplant-derived substances.

“β-glucan” means all β-glucan, including specific types of β-glucan,such as β-1,3-glucan or β-1,3;1,6-glucan. Moreover, β-1,3;1,6-glucan isa type of β-1,3-glucan. Therefore, the term “β-1,3-glucan” includesβ-1,3;1,6-glucan.

“Pectin” means any naturally-occurring oligosaccharide or polysaccharidethat comprises galacturonic acid that may be found in the cell wall of aplant. Different varieties and grades of pectin having varied physicaland chemical properties are known in the art. Indeed, the structure ofpectin can vary significantly between plants, between tissues, and evenwithin a single cell wall. Generally, pectin is made up of negativelycharged acidic sugars (galacturonic acid), and some of the acidic groupsare in the form of a methyl ester group. The degree of esterification ofpectin is a measure of the percentage of the carboxyl groups attached tothe galactopyranosyluronic acid units that are esterified with methanol.

Pectin having a degree of esterification of less than 50% (i.e., lessthan 50% of the carboxyl groups are methylated to form methyl estergroups) are classified as low-ester, low methoxyl, or low methylated(“LM”) pectins, while those having a degree of esterification of 50% orgreater (i.e., more than 50% of the carboxyl groups are methylated) areclassified as high-ester, high methoxyl or high methylated (“HM”)pectins. Very low (“VL”) pectins, a subset of low methylated pectins,have a degree of esterification that is less than approximately 15%.

As used herein, “lactoferrin from a non-human source” means lactoferrinwhich is produced by or obtained from a source other than human breastmilk. For example, lactoferrin for use in the present disclosureincludes human lactoferrin produced by a genetically modified organismas well as non-human lactoferrin. The term “organism”, as used herein,refers to any contiguous living system, such as animal, plant, fungus ormicro-organism.

As used herein, “non-human lactoferrin” means lactoferrin that has anamino acid sequence that is different than the amino acid sequence ofhuman lactoferrin.

“Pathogen” means an organism that causes a disease state or pathologicalsyndrome. Examples of pathogens may include bacteria, viruses,parasites, fungi, microbes or combination(s) thereof.

“Modulate” or “modulating” means exerting a modifying, controllingand/or regulating influence. In some embodiments, the term “modulating”means exhibiting an increasing or stimulatory effect on the level/amountof a particular component. In other embodiments, “modulating” meansexhibiting a decreasing or inhibitory effect on the level/amount of aparticular component.

All percentages, parts and ratios as used herein are by weight of thetotal formulation, unless otherwise specified.

All amounts specified as administered “per day” may be delivered in oneunit dose, in a single serving or in two or more doses or servingsadministered over the course of a 24 hour period.

The nutritional composition of the present disclosure may besubstantially free of any optional or selected ingredients describedherein, provided that the remaining nutritional composition stillcontains all of the required ingredients or features described herein.In this context, and unless otherwise specified, the term “substantiallyfree” means that the selected composition may contain less than afunctional amount of the optional ingredient, typically less than 0.1%by weight, and also, including zero percent by weight of such optionalor selected ingredient.

All references to singular characteristics or limitations of the presentdisclosure shall include the corresponding plural characteristic orlimitation, and vice versa, unless otherwise specified or clearlyimplied to the contrary by the context in which the reference is made.

All combinations of method or process steps as used herein can beperformed in any order, unless otherwise specified or clearly implied tothe contrary by the context in which the referenced combination is made.

The methods and compositions of the present disclosure, includingcomponents thereof, can comprise, consist of, or consist essentially ofthe essential elements and limitations of the embodiments describedherein, as well as any additional or optional ingredients, components orlimitations described herein or otherwise useful in nutritionalcompositions.

As used herein, the term “about” should be construed to refer to both ofthe numbers specified as the endpoint(s) of any range. Any reference toa range should be considered as providing support for any subset withinthat range.

The present disclosure is directed to nutritional compositionscomprising inositol, to uses thereof, and to methods comprisingadministration of those nutritional compositions to a pediatric or adultsubject. The nutritional compositions of the present disclosure supportand improve neurological health and development.

Inositol is transported across the blood-brain barrier by simplediffusion and a stereospecific saturation transport system. Moreover,the brain can take up inositol after exogenous administration. It hasthus been found that oral administration of inositol can engenderenhance neurological conditions for brain benefits.

It has been found that nutritional supplementation of inositolrepresents a feasible and effective approach to promote oligodendrocytesurvival and proliferation in a dose dependent manner, resulting in aconsistent increase in the number of oligodendrocyte precursor cells.Nutritional supplementation with inositol provides benefits for enhanceddevelopmental myelination by which it translates into a fundamentalbenefit for brain development. Given the importance of functionalmyelination, nutritional supplementation of inositol is beneficial topediatric and adult subjects by enhancing brain development and health.Because the nature and characteristics of inositol allow it to cross theblood brain barrier, inositol can be considered a novel brain nutrient,synergizing with other nutrients to provide comprehensive braindevelopment benefits. Moreover, the positive effects on enhanceddevelopmental myelination from inositol can be beneficial for preterminfants as well as those diagnosed with white matter diseases (such ascerebral palsy and periventricular leukomalacia). Inositol can also bebeneficial in other situations where myelination can be an issue, suchas with patients having multiple sclerosis and in post radiationsupplementation for promotion of recovery of OPCs. Moreover, the sweettaste of inositol provides further advantages in terms of palatabilityto consumers, especially infants and children.

In certain embodiments, inositol is present in the nutritionalcompositions of the present disclosure at a level of at least about 9mg/100 kcal; in other embodiments, inositol should be present at a levelof no greater than about 42 mg/100 kcal. In still other embodiments, thenutritional composition comprises inositol at a level of about 12 mg/100kcal to about 40 mg/100 kcal. In a further embodiment, inositol ispresent in the nutritional composition at a level of about 17 mg/100kcal to about 37 mg/100 kcal. Moreover, inositol can be present asexogenous inositol or inherent inositol. In embodiments, a majorfraction of the inositol (i.e., at least 40%) is exogenous inositol. Incertain embodiments, the ratio of exogenous to inherent inositol is atleast 50:50; in other embodiments, the ratio of exogenous to inherentinositol is at least 65:35. In still other embodiments, the ratio ofexogenous inositol to inherent inositol in the disclosed nutritionalcomposition is at least 75:25.

In some embodiments, the nutritional composition(s) of the disclosuremay also comprise at least one protein or protein equivalent source,which can be any used in the art, e.g., nonfat milk, whey protein,casein, soy protein, hydrolyzed protein, amino acids, and the like.Bovine milk protein sources useful in practicing the present disclosureinclude, but are not limited to, milk protein powders, milk proteinconcentrates, milk protein isolates, nonfat milk solids, nonfat milk,nonfat dry milk, whey protein, whey protein isolates, whey proteinconcentrates, sweet whey, acid whey, casein, acid casein, caseinate(e.g. sodium caseinate, sodium calcium caseinate, calcium caseinate) andany combinations thereof.

In some embodiments, the proteins of the nutritional composition areprovided as intact proteins. In other embodiments, the proteins areprovided as a combination of both intact proteins and hydrolyzedproteins. In certain embodiments, the proteins may be partiallyhydrolyzed or extensively hydrolyzed. In still other embodiments, theprotein equivalent source comprises amino acids. In yet anotherembodiment, the protein source may be supplemented withglutamine-containing peptides. In another embodiment, the proteincomponent comprises extensively hydrolyzed protein. In still anotherembodiment, the protein component of the nutritional compositionconsists essentially of extensively hydrolyzed protein in order tominimize the occurrence of food allergy. In yet another embodiment, theprotein source may be supplemented with glutamine-containing peptides.

Accordingly, in some embodiments, the protein component of thenutritional composition comprises either partially or extensivelyhydrolyzed protein, such as protein from cow's milk. The hydrolyzedproteins may be treated with enzymes to break down some or most of theproteins that cause adverse symptoms with the goal of reducing allergicreactions, intolerance, and sensitization. Moreover, the proteins may behydrolyzed by any method known in the art.

The terms “protein hydrolysates” or “hydrolyzed protein” are usedinterchangeably herein and refer to hydrolyzed proteins, wherein thedegree of hydrolysis is may be from about 20% to about 80%, or fromabout 30% to about 80%, or even from about 40% to about 60%.

When a peptide bond in a protein is broken by enzymatic hydrolysis, oneamino group is released for each peptide bond broken, causing anincrease in amino nitrogen. It should be noted that even non-hydrolyzedprotein would contain some exposed amino groups. Hydrolyzed proteinswill also have a different molecular weight distribution than thenon-hydrolyzed proteins from which they were formed. The functional andnutritional properties of hydrolyzed proteins can be affected by thedifferent size peptides. A molecular weight profile is usually given bylisting the percent by weight of particular ranges of molecular weight(in Daltons) fractions (e.g., 2,000 to 5,000 Daltons, greater than 5,000Daltons).

In a particular embodiment, the nutritional composition is protein-freeand contains free amino acids as a protein equivalent source. In thisembodiment, the amino acids may comprise, but are not limited to,histidine, isoleucine, leucine, lysine, methionine, cysteine,phenylalanine, tyrosine, threonine, tryptophan, valine, alanine,arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine,proline, serine, carnitine, taurine and mixtures thereof. In someembodiments, the amino acids may be branched chain amino acids. In otherembodiments, small amino acid peptides may be included as the proteincomponent of the nutritional composition. Such small amino acid peptidesmay be naturally occurring or synthesized. The amount of free aminoacids in the nutritional composition may vary from about 1 to about 5g/100 kcal. In an embodiment, 100% of the free amino acids have amolecular weight of less than 500 Daltons. In this embodiment, thenutritional formulation may be hypoallergenic.

In an embodiment, the protein source comprises from about 40% to about85% whey protein and from about 15% to about 60% casein.

In some embodiments, the nutritional composition comprises no greaterthan 7 g/100 kcal, and, in certain embodiments, between about 1 g andabout 7 g of a protein and/or protein equivalent source per 100 kcal. Inother embodiments, the nutritional composition comprises between about3.5 g and about 4.5 g of protein or protein equivalent per 100 kcal.

In some embodiments, the nutritional composition comprises at least onecarbohydrate source. Carbohydrate sources can be any used in the art,e.g., lactose, glucose, fructose, corn syrup solids, maltodextrins,sucrose, starch, rice syrup solids, and the like. The amount of theadditional carbohydrate component in the nutritional compositiontypically can be greater than 5 g/100 kcal; in some embodiments, it canvary from between about 5 g and about 25 g/100 kcal. In someembodiments, the amount of carbohydrate is between about 6 g and about22 g/100 kcal. In other embodiments, the amount of carbohydrate isbetween about 12 g and about 14 g/100 kcal. In some embodiments, cornsyrup solids are preferred. Moreover, hydrolyzed, partially hydrolyzed,and/or extensively hydrolyzed carbohydrates may be desirable forinclusion in the nutritional composition due to their easydigestibility. Specifically, hydrolyzed carbohydrates are less likely tocontain allergenic epitopes.

Non-limiting examples of carbohydrate materials suitable for use hereininclude hydrolyzed or intact, naturally or chemically modified, starchessourced from corn, tapioca, rice or potato, in waxy or non-waxy forms.Non-limiting examples of suitable carbohydrates include varioushydrolyzed starches characterized as hydrolyzed cornstarch,maltodextrin, maltose, corn syrup, dextrose, corn syrup solids, glucose,and various other glucose polymers and combinations thereof.Non-limiting examples of other suitable carbohydrates include thoseoften referred to as sucrose, lactose, fructose, high fructose cornsyrup, indigestible oligosaccharides such as fructooligosaccharides andcombinations thereof.

In one particular embodiment, the carbohydrate component of thenutritional composition is comprised of 100% lactose. In anotherembodiment, the additional carbohydrate component comprises betweenabout 0% and 60% lactose. In another embodiment, the carbohydratecomponent comprises between about 15% and 55% lactose. In yet anotherembodiment, the carbohydrate component comprises between about 20% and30% lactose. In these embodiments, the remaining source of carbohydratesmay be any carbohydrate known in the art. In an embodiment, thecarbohydrate component comprises about 25% lactose and about 75% cornsyrup solids.

In some embodiments, the carbohydrate may comprise at least one starchor starch component. A starch is a carbohydrate composed of two distinctpolymer fractions: amylose and amylopectin. Amylose is the linearfraction consisting of α-1,4 linked glucose units. Amylopectin has thesame structure as amylose, but some of the glucose units are combined inan α-1,6 linkage, giving rise to a branched structure. Starchesgenerally contain 17-24% amylose and from 76-83% amylopectin. Yetspecial genetic varieties of plants have been developed that producestarch with unusual amylose to amylopectin ratios. Some plants producestarch that is free of amylose. These mutants produce starch granules inthe endosperm and pollen that stain red with iodine and that containnearly 100% amylopectin. Predominant among such amylopectin producingplants are waxy corn, waxy sorghum and waxy rice starch.

The performance of starches under conditions of heat, shear and acid maybe modified or improved by chemical modifications. Modifications areusually attained by introduction of substituent chemical groups. Forexample, viscosity at high temperatures or high shear can be increasedor stabilized by cross-linking with di- or polyfunctional reagents, suchas phosphorus oxychloride.

In some instances, the nutritional compositions of the presentdisclosure comprise at least one starch that is gelatinized orpregelatinized. As is known in the art, gelatinization occurs whenpolymer molecules interact over a portion of their length to form anetwork that entraps solvent and/or solute molecules. Moreover, gelsform when pectin molecules lose some water of hydration owing tocompetitive hydration of cosolute molecules. Factors that influence theoccurrence of gelation include pH, concentration of cosolutes,concentration and type of cations, temperature and pectin concentration.Notably, LM pectin will gel only in the presence of divalent cations,such as calcium ions. And among LM pectins, those with the lowest degreeof esterification have the highest gelling temperatures and the greatestneed for divalent cations for crossbridging.

Meanwhile, pregelatinization of starch is a process of precooking starchto produce material that hydrates and swells in cold water. Theprecooked starch is then dried, for example by drum drying or spraydrying. Moreover the starch of the present disclosure can be chemicallymodified to further extend the range of its finished properties. Thenutritional compositions of the present disclosure may comprise at leastone pregelatinized starch.

Native starch granules are insoluble in water, but, when heated inwater, native starch granules begin to swell when sufficient heat energyis present to overcome the bonding forces of the starch molecules. Withcontinued heating, the granule swells to many times its original volume.The friction between these swollen granules is the major factor thatcontributes to starch paste viscosity.

The nutritional composition of the present disclosure may comprisenative or modified starches, such as, for example, waxy corn starch,waxy rice starch, corn starch, rice starch, potato starch, tapiocastarch, wheat starch or any mixture thereof. Generally, common cornstarch comprises about 25% amylose, while waxy corn starch is almosttotally made up of amylopectin. Meanwhile, potato starch generallycomprises about 20% amylose, rice starch comprises anamylose:amylopectin ratio of about 20:80, and waxy rice starch comprisesonly about 2% amylose. Further, tapioca starch generally comprises about15% to about 18% amylose, and wheat starch has an amylose content ofaround 25%.

In some embodiments, the nutritional composition comprises gelatinizedand/or pre-gelatinized waxy corn starch. In other embodiments, thenutritional composition comprises gelatinized and/or pre-gelatinizedtapioca starch. Other gelatinized or pre-gelatinized starches, such asrice starch or potato starch may also be used.

Suitable fats or lipids for use in the nutritional composition of thepresent disclosure may be any known or used in the art, including butnot limited to, animal sources, e.g., milk fat, butter, butter fat, eggyolk lipid; marine sources, such as fish oils, marine oils, single celloils; vegetable and plant oils, such as corn oil, canola oil, sunfloweroil, soybean oil, palmolein, coconut oil, high oleic sunflower oil,evening primrose oil, rapeseed oil, olive oil, flaxseed (linseed) oil,cottonseed oil, high oleic safflower oil, palm stearin, palm kernel oil,wheat germ oil; medium chain triglyceride oils and emulsions and estersof fatty acids; and any combinations thereof.

The amount of lipids or fats is, in one embodiment, no greater thanabout 7 g/100 kcal; in some embodiments, the lipid or fat is present ata level of from about 2 to about 7 g/100 kcal.

The nutritional composition may also contain one or more prebiotics(also referred to as a prebiotic component) in certain embodiments.Prebiotics exert health benefits, which may include, but are not limitedto, selective stimulation of the growth and/or activity of one or alimited number of beneficial gut bacteria, stimulation of the growthand/or activity of ingested probiotic microorganisms, selectivereduction in gut pathogens, and favorable influence on gut short chainfatty acid profile. Such prebiotics may be naturally-occurring,synthetic, or developed through the genetic manipulation of organismsand/or plants, whether such new source is now known or developed later.Prebiotics useful in the present disclosure may includeoligosaccharides, polysaccharides, and other prebiotics that containfructose, xylose, soya, galactose, glucose and mannose.

More specifically, prebiotics useful in the present disclosure mayinclude polydextrose (PDX), polydextrose powder, lactulose,lactosucrose, raffinose, gluco-oligosaccharide, inulin,fructo-oligosaccharide (FOS), isomalto-oligosaccharide, soybeanoligosaccharides, lactosucrose, xylo-oligosaccharide (XOS),chito-oligosaccharide, manno-oligosaccharide, aribino-oligosaccharide,siallyl-oligosaccharide, fuco-oligosaccharide, galacto-oligosaccharides(GOS) and gentio-oligosaccharides.

In an embodiment, the total amount of prebiotics present in thenutritional composition may be from about 1.0 g/L to about 10.0 g/L ofthe composition. More preferably, the total amount of prebiotics presentin the nutritional composition may be from about 2.0 g/L and about 8.0g/L of the composition. In some embodiments, the total amount ofprebiotics present in the nutritional composition may be from about 0.01g/100 kcal to about 1.5 g/100 kcal. In certain embodiments, the totalamount of prebiotics present in the nutritional composition may be fromabout 0.15 g/100 kcal to about 1.5 g/100 kcal. Moreover, the nutritionalcomposition may comprise a prebiotic component comprising PDX. In someembodiments, the prebiotic component comprises at least 20% w/w PDX, GOSor a mixture thereof.

The amount of PDX in the nutritional composition may, in an embodiment,be within the range of from about 0.015 g/100 kcal to about 1.5 g/100kcal. In another embodiment, the amount of polydextrose is within therange of from about 0.2 g/100 kcal to about 0.6 g/100 kcal. In someembodiments, PDX may be included in the nutritional composition in anamount sufficient to provide between about 1.0 g/L and 10.0 g/L. Inanother embodiment, the nutritional composition contains an amount ofPDX that is between about 2.0 g/L and 8.0 g/L. And in still otherembodiments, the amount of PDX in the nutritional composition may befrom about 0.05 g/100 kcal to about 1.5 g/100 kcal.

The prebiotic component also comprises GOS in some embodiments. Theamount of GOS in the nutritional composition may, in an embodiment, befrom about 0.015 g/100 kcal to about 1.0 g/100 kcal. In anotherembodiment, the amount of GOS in the nutritional composition may be fromabout 0.2 g/100 kcal to about 0.5 g/100 kcal.

In a particular embodiment of the present disclosure, PDX isadministered in combination with GOS.

In a particular embodiment, GOS and PDX are supplemented into thenutritional composition in a total amount of at least about 0.015 g/100kcal or about 0.015 g/100 kcal to about 1.5 mg/100 kcal. In someembodiments, the nutritional composition may comprise GOS and PDX in atotal amount of from about 0.1 to about 1.0 mg/100 kcal.

Lactoferrin can also included in some embodiments of the nutritionalcomposition of the present disclosure. Lactoferrins are single chainpolypeptides of about 80 kD containing 1-4 glycans, depending on thespecies. The 3-D structures of lactoferrin of different species are verysimilar, but not identical. Each lactoferrin comprises two homologouslobes, called the N- and C-lobes, referring to the N-terminal andC-terminal part of the molecule, respectively. Each lobe furtherconsists of two sub-lobes or domains, which form a cleft where theferric ion (Fe³⁺) is tightly bound in synergistic cooperation with a(bi)carbonate anion. These domains are called N1, N2, C1 and C2,respectively. The N-terminus of lactoferrin has strong cationic peptideregions that are responsible for a number of important bindingcharacteristics. Lactoferrin has a very high isoelectric point (˜pI 9)and its cationic nature plays a major role in its ability to defendagainst bacterial, viral, and fungal pathogens. There are severalclusters of cationic amino acids residues within the N-terminal regionof lactoferrin mediating the biological activities of lactoferrinagainst a wide range of microorganisms. For instance, the N-terminalresidues 1-47 of human lactoferrin (1-48 of bovine lactoferrin) arecritical to the iron-independent biological activities of lactoferrin.In human lactoferrin, residues 2 to 5 (RRRR) and 28 to 31 (RKVR) arearginine-rich cationic domains in the N-terminus especially critical tothe antimicrobial activities of lactoferrin. A similar region in theN-terminus is found in bovine lactoferrin (residues 17 to 42;FKCRRWQWRMKKLGAPSITCVRRAFA).

Lactoferrins from different host species may vary in their amino acidsequences though commonly possess a relatively high isoelectric pointwith positively charged amino acids at the end terminal region of theinternal lobe. Suitable non-human lactoferrins for use in the presentdisclosure include, but are not limited to, those having at least 48%homology with the amino acid sequence of human lactoferrin. Forinstance, bovine lactoferrin (“bLF”) has an amino acid composition whichhas about 70% sequence homology to that of human lactoferrin. In someembodiments, the non-human lactoferrin has at least 55% homology withhuman lactoferrin and in some embodiments, at least 65% homology.Non-human lactoferrins acceptable for use in the present disclosureinclude, without limitation, bLF, porcine lactoferrin, equinelactoferrin, buffalo lactoferrin, goat lactoferrin, murine lactoferrinand camel lactoferrin.

In one embodiment, lactoferrin is present in the nutritional compositionin an amount of at least about 15 mg/100 kCal. In certain embodiments,the nutritional composition may include between about 15 and about 300mg lactoferrin per 100 kCal. In another embodiment, where thenutritional composition is an infant formula, the nutritionalcomposition may comprise lactoferrin in an amount of from about 60 mg toabout 150 mg lactoferrin per 100 kCal; in yet another embodiment, thenutritional composition may comprise about 60 mg to about 100 mglactoferrin per 100 kCal.

In some embodiments, the nutritional composition can include lactoferrinin the quantities of from about 0.5 mg to about 1.5 mg per milliliter offormula. In nutritional compositions replacing human milk, lactoferrinmay be present in quantities of from about 0.6 mg to about 1.3 mg permilliliter of formula. In certain embodiments, the nutritionalcomposition may comprise between about 0.1 and about 2 grams lactoferrinper liter. In some embodiments, the nutritional composition includesbetween about 0.6 and about 1.5 grams lactoferrin per liter of formula.

The bLF that is used in certain embodiments may be any bLF isolated fromwhole milk and/or having a low somatic cell count, wherein “low somaticcell count” refers to a somatic cell count less than 200,000 cells/mL.By way of example, suitable bLF is available from Tatua Co-operativeDairy Co. Ltd., in Morrinsville, New Zealand, from FrieslandCampina Domoin Amersfoort, Netherlands or from Fonterra Co-Operative Group Limitedin Auckland, New Zealand.

Lactoferrin for use in the present disclosure may be, for example,isolated from the milk of a non-human animal or produced by agenetically modified organism. For example, in U.S. Pat. No. 4,791,193,incorporated by reference herein in its entirety, Okonogi et al.discloses a process for producing bovine lactoferrin in high purity.Generally, the process as disclosed includes three steps. Raw milkmaterial is first contacted with a weakly acidic cationic exchanger toabsorb lactoferrin followed by the second step where washing takes placeto remove nonabsorbed substances. A desorbing step follows wherelactoferrin is removed to produce purified bovine lactoferrin. Othermethods may include steps as described in U.S. Pat. Nos. 7,368,141,5,849,885, 5,919,913 and 5,861,491, the disclosures of which are allincorporated by reference in their entirety.

In certain embodiments, lactoferrin utilized in the present disclosuremay be provided by an expanded bed absorption (“EBA”) process forisolating proteins from milk sources. EBA, also sometimes calledstabilized fluid bed adsorption, is a process for isolating a milkprotein, such as lactoferrin, from a milk source comprises establishingan expanded bed adsorption column comprising a particulate matrix,applying a milk source to the matrix, and eluting the lactoferrin fromthe matrix with an elution buffer comprising about 0.3 to about 2.0 Msodium chloride. Any mammalian milk source may be used in the presentprocesses, although in particular embodiments, the milk source is abovine milk source. The milk source comprises, in some embodiments,whole milk, reduced fat milk, skim milk, whey, casein, or mixturesthereof.

In particular embodiments, the target protein is lactoferrin, thoughother milk proteins, such as lactoperoxidases or lactalbumins, also maybe isolated. In some embodiments, the process comprises the steps ofestablishing an expanded bed adsorption column comprising a particulatematrix, applying a milk source to the matrix, and eluting thelactoferrin from the matrix with about 0.3 to about 2.0M sodiumchloride. In other embodiments, the lactoferrin is eluted with about 0.5to about 1.0 M sodium chloride, while in further embodiments, thelactoferrin is eluted with about 0.7 to about 0.9 M sodium chloride.

The expanded bed adsorption column can be any known in the art, such asthose described in U.S. Pat. Nos. 7,812,138, 6,620,326, and 6,977,046,the disclosures of which are hereby incorporated by reference herein. Insome embodiments, a milk source is applied to the column in an expandedmode, and the elution is performed in either expanded or packed mode. Inparticular embodiments, the elution is performed in an expanded mode.For example, the expansion ratio in the expanded mode may be about 1 toabout 3, or about 1.3 to about 1.7. EBA technology is further describedin international published application nos. WO 92/00799, WO 02/18237, WO97/17132, which are hereby incorporated by reference in theirentireties.

The isoelectric point of lactoferrin is approximately 8.9. Prior EBAmethods of isolating lactoferrin use 200 mM sodium hydroxide as anelution buffer. Thus, the pH of the system rises to over 12, and thestructure and bioactivity of lactoferrin may be comprised, byirreversible structural changes. It has now been discovered that asodium chloride solution can be used as an elution buffer in theisolation of lactoferrin from the EBA matrix. In certain embodiments,the sodium chloride has a concentration of about 0.3 M to about 2.0 M.In other embodiments, the lactoferrin elution buffer has a sodiumchloride concentration of about 0.3 M to about 1.5 M, or about 0.5 m toabout 1.0 M.

The nutritional composition of the disclosure can also contain a sourceof LCPUFAs in certain embodiments; especially a source of LCPUFAs thatcomprises DHA. Other suitable LCPUFAs include, but are not limited to,α-linoleic acid, γ-linoleic acid, linoleic acid, linolenic acid,eicosapentaenoic acid (EPA) and ARA. Indeed, DHA and/or ARA may actsynergistically with inositol to further improve neurological health anddevelopment.

In an embodiment, especially if the nutritional composition is an infantformula, the nutritional composition is supplemented with both DHA andARA. In this embodiment, the weight ratio of ARA:DHA may be betweenabout 1:3 and about 9:1. In a particular embodiment, the ratio ofARA:DHA is from about 1:2 to about 4:1.

The amount of long chain polyunsaturated fatty acid in the nutritionalcomposition is advantageously at least about 5 mg/100 kcal, and may varyfrom about 5 mg/100 kcal to about 100 mg/100 kcal, more preferably fromabout 10 mg/100 kcal to about 50 mg/100 kcal.

The nutritional composition may be supplemented with oils containing DHAand/or ARA using standard techniques known in the art. For example, DHAand ARA may be added to the composition by replacing an equivalentamount of an oil, such as high oleic sunflower oil, normally present inthe composition. As another example, the oils containing DHA and ARA maybe added to the composition by replacing an equivalent amount of therest of the overall fat blend normally present in the compositionwithout DHA and ARA.

If utilized, the source of DHA and/or ARA may be any source known in theart such as marine oil, fish oil, single cell oil, egg yolk lipid, andbrain lipid. In some embodiments, the DHA and ARA are sourced fromsingle cell Martek oils, DHASCO® and ARASCO®, or variations thereof. TheDHA and ARA can be in natural form, provided that the remainder of theLCPUFA source does not result in any substantial deleterious effect onthe infant. Alternatively, the DHA and ARA can be used in refined form.

In an embodiment, sources of DHA and ARA are single cell oils as taughtin U.S. Pat. Nos. 5,374,567; 5,550,156; and 5,397,591, the disclosuresof which are incorporated herein in their entirety by reference.However, the present disclosure is not limited to only such oils.

In some embodiments the nutritional composition may include an enrichedlipid fraction derived from milk. The enriched lipid fraction derivedfrom milk may be produced by any number of fractionation techniques.These techniques include but are not limited to melting pointfractionation, organic solvent fractionation, super critical fluidfractionation, and any variants and combinations thereof. In someembodiments the nutritional composition may include an enriched lipidfraction derived from milk that contains milk fat globules.

In certain embodiments, the addition of the enriched lipid fraction orthe enriched lipid fraction including milk fat globules may provide asource of saturated fatty acids, trans-fatty acids, monounsaturatedfatty acids, polyunsaturated fatty acids, OBCFAs, BCFAs, CLA,cholesterol, phospholipids, and/or milk fat globule membrane proteins tothe nutritional composition.

The milk fat globules may have an average diameter (volume-surface areaaverage diameter) of at least about 2 μm. In some embodiments, theaverage diameter is in the range of from about 2 μm to about 13 μm. Inother embodiments, the milk fat globules may range from about 2.5 μm toabout 10 μm. Still in other embodiments, the milk fat globules may rangein average diameter from about 3 μm to about 6 μm. The specific surfacearea of the globules is, in certain embodiments, less than 3.5 m²/g, andin other embodiments is between about 0.9 m²/g to about 3 m²/g. Withoutbeing bound by any particular theory, it is believed that milk fatglobules of the aforementioned sizes are more accessible to lipasestherefore leading to better digestion lipid digestion.

In some embodiments the enriched lipid fraction and/or milk fat globulescontain saturated fatty acids. The saturated fatty acids may be presentin a concentration from about 0.1 g/100 kcal to about 8.0 g/100 kcal. Incertain embodiments the saturated fatty acids may be present from about0.5 g/100 kcal to about 2.0 g/100 kcal. In still other embodiments thesaturated fatty acids may be present from about 3.5 g/100 kcal to about6.9 g/100 kcal.

Examples of saturated fatty acids suitable for inclusion include, butare not limited to, butyric, valeric, caproic, caprylic, decanoic,lauric, myristic, palmitic, steraic, arachidic, behenic, alignoceric,tetradecanoic, hexadecanoic, palmitic, and octadecanoic acid, and/orcombinations and mixtures thereof.

Additionally, the enriched lipid fraction and/or milk fat globules maycomprise, in some embodiments, lauric acid. Lauric acid, also known asdodecanoic acid, is a saturated fatty acid with a 12-carbon atom chainand is believed to be one of the main antiviral and antibacterialsubstances currently found in human breast milk. The milk fat globulesmay be enriched with triglycerides containing lauric acid at either theSn-1, Sn-2 and/or Sn-3 positions. Without being bound by any particulartheory, it is believed that when the enriched lipid fraction isingested, the mouth lingual lipase and pancreatic lipase will hydrolyzethe triglycerides to a mixture of glycerides including mono-lauric andfree lauric acid.

The concentration of lauric acid in the globules varies from 80 mg/100ml to 800 mg/100 ml. The concentration of monolauryl n the globules canbe in the range of 20 mg/100 ml to 300 mg/100 ml feed. In someembodiments, the range is 60 mg/100 ml to 130 mg/100 ml.

The enriched lipid fraction and/or milk fat globules may containtrans-fatty acids in certain embodiments. The trans-fatty acids includedin the milk fat globules may be monounsaturated or polyunsaturatedtrans-fatty acids. In some embodiments the trans-fatty acids may bepresent in an amount from about 0.2 g/100 kcal to about 7.0 g/100 kcal.In other embodiments the trans-fatty acids may be present in an amountfrom about 3.4 g/100 kcal to about 5.2 g/100 kcal. In yet otherembodiments the trans-fatty acids may be present from about 1.2 g/100kcal to about 4.3 g/100 kcal.

Examples of trans-fatty acids for inclusion include, but are not limitedto, vaccenic, or elaidic acid, and mixtures thereof. Moreover, whenconsumed, mammals convert vaccenic acid into rumenic acid, which is aconjugated linoleic acid that exhibits anticarcinogenic properties.Additionally, a diet enriched with vaccenic acid may help lower totalcholesterol, LDL cholesterol and triglyceride levels.

In some embodiments the enriched lipid fraction and/or milk fat globulesmay contain OBCFAs. In certain embodiments, the OBCFAs may be present inan amount from about 0.3 g/100 kcal to about 6.1 g/100 kcal. In otherembodiments OBCFAs may be present in an amount from about 2.2 g/100 kcalto about 4.3 g/100 kcal. In yet another embodiment OBCFAs may be presentin an amount from about 3.5 g/100 kcal to about 5.7 g/100 kcal. In stillother embodiments, the milk fat globules comprise at least one OBCFA.

Typically, an infant may absorb OBCFAs while in utero and from thebreast milk of a nursing mother. Therefore, OBCFAs that are identifiedin human milk are preferred for inclusion in the milk fat globules ofthe nutritional composition. Addition of OBCFAs to infant or children'sformulas allows such formulas to mirror the composition andfunctionality of human milk and to promote general health andwell-being.

In some embodiments, the enriched lipid fraction and/or milk fatglobules may comprise BCFAs. In some embodiments the BCFAs are presentat a concentration from about 0.2 g/100 kcal and about 5.82 g/100 kcal.In another embodiment, the BCFAs are present in an amount of from about2.3 g/100 kcal to about 4.2 g/100 kcal. In yet another embodiment theBCFAs are present from about 4.2 g/100 kcal to about 5.82 g/100 kcal. Instill other embodiments, the milk fat globules comprise at least oneBCFA.

BCFAs that are identified in human milk are preferred for inclusion inthe nutritional composition. Addition of BCFAs to infant or children'sformulas allows such formulas to mirror the composition andfunctionality of human milk and to promote general health andwell-being.

In certain embodiments the enriched lipid fraction and/or milk fatglobules may comprise CLA. In some embodiments CLA may be present in aconcentration from about 0.4 g/100 kcal to about 2.5 g/100 kcal. Inother embodiments CLA may be present from about 0.8 g/100 kcal to about1.2 g/100 kcal. In yet other embodiments CLA may be present from about1.2 g/100 kcal to about 2.3 g/100 kcal. In still other embodiments, themilk fat globules comprise at least one CLA.

CLAs that are identified in human milk are preferred for inclusion inthe nutritional composition. Typically, CLAs are absorbed by the infantfrom the human milk of a nursing mother. Addition of CLAs to infant orchildren's formulas allows such formulas to mirror the composition andfunctionality of human milk and to promote general health and wellbeing.

Examples of CLAs found in the milk fat globules for the nutritionalcomposition include, but are not limited to, cis-9, trans-11 CLA,trans-10, cis-12 CLA, cis-9, trans-12 octadecadienoic acid, and mixturesthereof.

The enriched lipid fraction and/or milk fat globules of the presentdisclosure comprise monounsaturated fatty acids in some embodiments. Theenriched lipid fraction and/or milk fat globules may be formulated toinclude monounsaturated fatty acids from about 0.8 g/100 kcal to about2.5 g/100 kcal. In other embodiments the milk fat globules may includemonounsaturated fatty acids from about 1.2 g/100 kcal to about 1.8 g/100kcal.

Examples of monounsaturated fatty acids suitable include, but are notlimited to, palmitoleic acid, cis-vaccenic acid, oleic acid, andmixtures thereof.

In certain embodiments, the enriched lipid fraction and/or milk fatglobules of the present disclosure comprise polyunsaturated fatty acidsfrom about 2.3 g/100 kcal to about 4.4 g/100 kcal. In other embodiments,the polyunsaturated fatty acids are present from about 2.7 g/100 kcal toabout 3.5 g/100 kcal. In yet another embodiment, the polyunsaturatedfatty acids are present from about 2.4 g/100 kcal to about 3.3 g/100kcal.

In some embodiments, the enriched lipid fraction and/or milk fatglobules of the present disclosure comprise polyunsaturated fatty acids,such as, for example linoleic acid, linolenic acid, octadecatrienoicacid, arachidonic acid (ARA), eicosatetraenoic acid, eicopsapentaenoicacid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA).Polyunsaturated fatty acids are the precursors for prostaglandins andeicosanoids, which are known to provide numerous health benefits,including, anti-inflammatory response, cholesterol absorption, andincreased bronchial function.

The enriched lipid fraction and/or milk fat globules of the presentdisclosure can also comprise cholesterol in some embodiments from about100 mg/100 kcal to about 400 mg/100 kcal. In another embodiment,cholesterol is present from about 200 mg/100 kcal to about 300 mg/100kcal. As is similar to human milk and bovine milk, the cholesterolincluded in the milk fat globules may be present in the outer bilayermembrane of the milk fat globule to provide stability to the globularmembrane.

In some embodiments, the enriched lipid fraction and/or milk fatglobules of the present disclosure comprise phospholipids from about 50mg/100 kcal to about 200 mg/100 kcal. In other embodiments, thephospholipids are present from about 75 mg/100 kcal to about 150 mg/100kcal. In yet other embodiments, the phospholipids are present at aconcentration of from about 100 mg/100 kcal to about 250 mg/100 kcal.

In certain embodiments, phospholipids may be incorporated into the milkfat globules to stabilize the milk fat globule by providing aphospholipid membrane or bilayer phospholipid membrane. Therefore, insome embodiments the milk fat globules may be formulated with higheramounts of phospholipids than those found in human milk.

The phospholipid composition of human milk lipids, as the weight percentof total phospholipids, is phosphatidylcholine (“PC”) 24.9%,phosphatidylethanolamine (“PE”) 27.7%, phosphatidylserine (“PS”) 9.3%,phosphatidylinositol (“PI”) 5.4%, and sphingomyelin (“SPM”) 32.4%,(Harzer, G. et al., Am. J. Clin. Nutr., Vol. 37, pp. 612-621 (1983)).Thus in one embodiment, the milk fat globules comprise one or more ofPC, PE, PS, PI, SPM, and mixtures thereof. Further, the phospholipidcomposition included in the milk fat globules may be formulated toprovide certain health benefits by incorporating desired phospholipids.

In certain embodiments, the enriched lipid fraction and/or milk fatglobules of the present disclosure comprise milk fat globule membraneprotein. In some embodiments, the milk fat globule membrane protein ispresent from about 50 mg/100 kcal to about 500 mg/100 kcal.

Galactolipids may be included, in some embodiments, in the enrichedlipid fraction and/or milk fat globules of the present disclosure. Forpurposes of this disclosure “galactolipids” refer to any glycolipidwhose sugar group is galactose. More specifically, galactolipids differfrom glycosphingolipids in that they do not have nitrogen in theircomposition. Galactolipids play an important role in supporting braindevelopment and overall neuronal health. Additionally, thegalactolipids, galactocerebroside and sulfatides constitute about 23%and 4% of total myelin lipid content respectively, and thus may beincorporated into the milk fat globules in some embodiments.

Additionally, the nutritional compositions of the present disclosurecomprise at least one source of pectin. The source of pectin maycomprise any variety or grade of pectin known in the art. In someembodiments, the pectin has a degree of esterification of less than 50%and is classified as low methylated (“LM”) pectin. In some embodiments,the pectin has a degree of esterification of greater than or equal to50% and is classified as high-ester or high methylated (“HM”) pectin. Instill other embodiments, the pectin is very low (“VL”) pectin, which hasa degree of esterification that is less than approximately 15%. Further,the nutritional composition of the present disclosure may comprise LMpectin, HM pectin, VL pectin, or any mixture thereof. The nutritionalcomposition may include pectin that is soluble in water. And, as knownin the art, the solubility and viscosity of a pectin solution arerelated to the molecular weight, degree of esterification, concentrationof the pectin preparation and the pH and presence of counterions.

Moreover, pectin has a unique ability to form gels. Generally, undersimilar conditions, a pectin's degree of gelation, the gellingtemperature, and the gel strength are proportional to one another, andeach is generally proportional to the molecular weight of the pectin andinversely proportional to the degree of esterification. For example, asthe pH of a pectin solution is lowered, ionization of the carboxylategroups is repressed, and, as a result of losing their charge, saccharidemolecules do not repel each other over their entire length. Accordingly,the polysaccharide molecules can associate over a portion of theirlength to form a gel. Yet pectins with increasing degrees of methylationwill gel at somewhat higher pH because they have fewer carboxylateanions at any given pH. (J. N. Bemiller, An Introduction to Pectins:Structure and Properties, Chemistry and Function of Pectins; Chapter 1;1986.)

The nutritional composition may comprise a gelatinized and/orpregelatinized starch together with pectin and/or gelatinized pectin.While not wishing to be bound by this or any other theory, it isbelieved that the use of pectin, such as LM pectin, which is ahydrocolloid of large molecular weight, together with starch granules,provides a synergistic effect that increases the molecular internalfriction within a fluid matrix. The carboxylic groups of the pectin mayalso interact with calcium ions present in the nutritional composition,thus leading to an increase in viscosity, as the carboxylic groups ofthe pectin form a weak gel structure with the calcium ion(s), and alsowith peptides present in the nutritional composition. In someembodiments, the nutritional composition comprises a ratio of starch topectin that is between about 12:1 and 20:1, respectively. In otherembodiments, the ratio of starch to pectin is about 17:1. In someembodiments, the nutritional composition may comprise between about 0.05and about 2.0% w/w pectin. In a particular embodiment, the nutritionalcomposition may comprise about 0.5% w/w pectin.

Pectins for use herein typically have a peak molecular weight of 8,000Daltons or greater. The pectins of the present disclosure have apreferred peak molecular weight of between 8,000 and about 500,000, morepreferred is between about 10,000 and about 200,000 and most preferredis between about 15,000 and about 100,000 Daltons. In some embodiments,the pectin of the present disclosure may be hydrolyzed pectin. Incertain embodiments, the nutritional composition comprises hydrolyzedpectin having a molecular weight less than that of intact or unmodifiedpectin. The hydrolyzed pectin of the present disclosure can be preparedby any means known in the art to reduce molecular weight. Examples ofsaid means are chemical hydrolysis, enzymatic hydrolysis and mechanicalshear. A preferred means of reducing the molecular weight is by alkalineor neutral hydrolysis at elevated temperature. In some embodiments, thenutritional composition comprises partially hydrolyzed pectin. Incertain embodiments, the partially hydrolyzed pectin has a molecularweight that is less than that of intact or unmodified pectin but morethan 3,300 Daltons.

The nutritional composition may contain at least one acidicpolysaccharide. An acidic polysaccharide, such as negatively chargedpectin, may induce an anti-adhesive effect on pathogens in a subject'sgastrointestinal tract. Indeed, nonhuman milk acidic oligosaccharidesderived from pectin are able to interact with the epithelial surface andare known to inhibit the adhesion of pathogens on the epithelialsurface.

In some embodiments, the nutritional composition comprises at least onepectin-derived acidic oligosaccharide. Pectin-derived acidicoligosaccharide(s) (pAOS) result from enzymatic pectinolysis, and thesize of a pAOS depends on the enzyme use and on the duration of thereaction. In such embodiments, the pAOS may beneficially affect asubject's stool viscosity, stool frequency, stool pH and/or feedingtolerance. The nutritional composition of the present disclosure maycomprise between about 2 g pAOS per liter of formula and about 6 g pAOSper liter of formula. In an embodiment, the nutritional compositioncomprises about 0.2 g pAOS/dL, corresponding to the concentration ofacidic oligosaccharides in human milk. (Fanaro et al., “AcidicOligosaccharides from Pectin Hydrolysate as New Component for InfantFormulae: Effect on Intestinal Flora, Stool Characteristics, and pH”,Journal of Pediatric Gastroenterology and Nutrition, 41: 186-190, August2005)

In some embodiments, the nutritional composition comprises up to about20% w/w of a mixture of starch and pectin. In some embodiments, thenutritional composition comprises up to about 19% starch and up to about1% pectin. In other embodiments, the nutritional composition comprisesabout up to about 15% starch and up to about 5% pectin. In still otherembodiments, the nutritional composition comprises up to about 18%starch and up to about 2% pectin. In some embodiments the nutritionalcomposition comprises between about 0.05% w/w and about 20% w/w of amixture of starch and pectin. Other embodiments include between about0.05% and about 19% w/w starch and between about 0.05% and about 1% w/wpectin. Further, the nutritional composition may comprise between about0.05% and about 15% w/w starch and between about 0.05% and about 5% w/wpectin.

In some embodiments the nutritional composition comprises sialic acid.Sialic acids are a family of over 50 members of 9-carbon sugars, all ofwhich are derivatives of neuroaminic acid. The predominant sialic acidfamily found in humans is from the N-acetylneuraminic acid sub-family.Sialic acids are found in milk, such as bovine and caprine. In mammals,neuronal cell membranes have the highest concentration of sialic acidcompared to other body cell membranes. Sialic acid residues are alsocomponents of gangliosides.

If included in the nutritional composition, sialic acid may be presentin an amount from about 0.5 mg/100 kcals to about 45 mg/100 kcal. Insome embodiments sialic acid may be present in an amount from about 5mg/100 kcals to about 30 mg/100 kcals. In still other embodiments,sialic acid may be present in an amount from about 10 mg/100 kcals toabout 25 mg/100 kcals.

In one embodiment, the nutritional composition may contain one or moreprobiotics. Any probiotic known in the art may be acceptable in thisembodiment. In a particular embodiment, the probiotic may be selectedfrom any Lactobacillus species, Lactobacillus rhamnosus GG (LGG) (ATCCnumber 53103), Bifidobacterium species, Bifidobacterium longum BB536(BL999, ATCC: BAA-999), Bifidobacterium longum AH1206 (NCIMB: 41382),Bifidobacterium breve AH1205 (NCIMB: 41387), Bifidobacterium infantis35624 (NCIMB: 41003), and Bifidobacterium animalis subsp. lactis BB-12(DSM No. 10140) or any combination thereof.

If included in the composition, the amount of the probiotic may varyfrom about 1×10⁴ to about 1.5×10¹² cfu of probiotic(s) per 100 kcal. Insome embodiments the amount of probiotic may be from about 1×10⁶ toabout 1×10⁹ cfu of probiotic(s) per 100 kcal. In certain otherembodiments the amount of probitic may vary from about 1×10⁷ cfu/100kcal to about 1×10⁸ cfu of probiotic(s) per 100 kcal.

In an embodiment, the probiotic(s) may be viable or non-viable. As usedherein, the term “viable”, refers to live microorganisms. The term“non-viable” or “non-viable probiotic” means non-living probioticmicroorganisms, their cellular components and/or metabolites thereof.Such non-viable probiotics may have been heat-killed or otherwiseinactivated, but they retain the ability to favorably influence thehealth of the host. The probiotics useful in the present disclosure maybe naturally-occurring, synthetic or developed through the geneticmanipulation of organisms, whether such source is now known or laterdeveloped.

In some embodiments, the nutritional composition may include a sourcecomprising probiotic cell equivalents, which refers to the level ofnon-viable, non-replicating probiotics equivalent to an equal number ofviable cells. The term “non-replicating” is to be understood as theamount of non-replicating microorganisms obtained from the same amountof replicating bacteria (cfu/g), including inactivated probiotics,fragments of DNA, cell wall or cytoplasmic compounds. In other words,the quantity of non-living, non-replicating organisms is expressed interms of cfu as if all the microorganisms were alive, regardless whetherthey are dead, non-replicating, inactivated, fragmented etc. Innon-viable probiotics are included in the nutritional composition, theamount of the probiotic cell equivalents may vary from about 1×10⁴ toabout 1.5×10¹⁰ cell equivalents of probiotic(s) per 100 kcal. In someembodiments the amount of probiotic cell equivalents may be from about1×10⁶ to about 1×10⁹ cell equivalents of probiotic(s) per 100 kcalnutritional composition. In certain other embodiments the amount ofprobiotic cell equivalents may vary from about 1×10⁷ to about 1×10⁸ cellequivalents of probiotic(s) per 100 kcal of nutritional composition.

In some embodiments, the probiotic source incorporated into thenutritional composition may comprise both viable colony-forming units,and non-viable cell-equivalents.

In some embodiments, the nutritional composition includes a culturesupernatant from a late-exponential growth phase of a probioticbatch-cultivation process. Without wishing to be bound by theory, it isbelieved that the activity of the culture supernatant can be attributedto the mixture of components (including proteinaceous materials, andpossibly including (exo)polysaccharide materials) as found released intothe culture medium at a late stage of the exponential (or “log”) phaseof batch cultivation of the probiotic. The term “culture supernatant” asused herein, includes the mixture of components found in the culturemedium. The stages recognized in batch cultivation of bacteria are knownto the skilled person. These are the “lag,” the “log” (“logarithmic” or“exponential”), the “stationary” and the “death” (or “logarithmicdecline”) phases. In all phases during which live bacteria are present,the bacteria metabolize nutrients from the media, and secrete (exert,release) materials into the culture medium. The composition of thesecreted material at a given point in time of the growth stages is notgenerally predictable.

In an embodiment, a culture supernatant is obtainable by a processcomprising the steps of (a) subjecting a probiotic such as LGG tocultivation in a suitable culture medium using a batch process; (b)harvesting the culture supernatant at a late exponential growth phase ofthe cultivation step, which phase is defined with reference to thesecond half of the time between the lag phase and the stationary phaseof the batch-cultivation process; (c) optionally removing low molecularweight constituents from the supernatant so as to retain molecularweight constituents above 5-6 kiloDaltons (kDa); (d) removing liquidcontents from the culture supernatant so as to obtain the composition.

The culture supernatant may comprise secreted materials that areharvested from a late exponential phase. The late exponential phaseoccurs in time after the mid exponential phase (which is halftime of theduration of the exponential phase, hence the reference to the lateexponential phase as being the second half of the time between the lagphase and the stationary phase). In particular, the term “lateexponential phase” is used herein with reference to the latter quarterportion of the time between the lag phase and the stationary phase ofthe LGG batch-cultivation process. In some embodiments, the culturesupernatant is harvested at a point in time of 75% to 85% of theduration of the exponential phase, and may be harvested at about ⅚ ofthe time elapsed in the exponential phase.

As noted, the disclosed nutritional composition may comprise a source ofβ-glucan. Glucans are polysaccharides, specifically polymers of glucose,which are naturally occurring and may be found in cell walls ofbacteria, yeast, fungi, and plants. Beta glucans (β-glucans) arethemselves a diverse subset of glucose polymers, which are made up ofchains of glucose monomers linked together via beta-type glycosidicbonds to form complex carbohydrates.

β-1,3-glucans are carbohydrate polymers purified from, for example,yeast, mushroom, bacteria, algae, or cereals. (Stone B A, Clarke A E.Chemistry and Biology of (1-3)-Beta-Glucans. London:Portland Press Ltd;1993.) The chemical structure of β-1,3-glucan depends on the source ofthe β-1,3-glucan. Moreover, various physiochemical parameters, such assolubility, primary structure, molecular weight, and branching, play arole in biological activities of β-1,3-glucans. (Yadomae T., Structureand biological activities of fungal beta-1,3-glucans. Yakugaku Zasshi.2000; 120:413-431.)

β-1,3-glucans are naturally occurring polysaccharides, with or withoutβ-1,6-glucose side chains that are found in the cell walls of a varietyof plants, yeasts, fungi and bacteria. β-1,3;1,6-glucans are thosecontaining glucose units with (1,3) links having side chains attached atthe (1,6) position(s). β-1,3;1,6 glucans are a heterogeneous group ofglucose polymers that share structural commonalities, including abackbone of straight chain glucose units linked by a β-1,3 bond withβ-1,6-linked glucose branches extending from this backbone. While thisis the basic structure for the presently described class of β-glucans,some variations may exist. For example, certain yeast β-glucans haveadditional regions of β(1,3) branching extending from the β(1,6)branches, which add further complexity to their respective structures.

β-glucans derived from baker's yeast, Saccharomyces cerevisiae, are madeup of chains of D-glucose molecules connected at the 1 and 3 positions,having side chains of glucose attached at the 1 and 6 positions.Yeast-derived β-glucan is an insoluble, fiber-like, complex sugar havingthe general structure of a linear chain of glucose units with a β-1,3backbone interspersed with β-1,6 side chains that are generally 6-8glucose units in length. More specifically, β-glucan derived frombaker's yeast is poly-(1,6)-β-D-glucopyranosyl-(1,3)-β-D-glucopyranose.

Furthermore, β-glucans are well tolerated and do not produce or causeexcess gas, abdominal distension, bloating or diarrhea in pediatricsubjects. Addition of β-glucan to a nutritional composition for apediatric subject, such as an infant formula, a growing-up milk oranother children's nutritional product, will improve the subject'simmune response by increasing resistance against invading pathogens andtherefore maintaining or improving overall health.

The nutritional composition of the present disclosure comprisesβ-glucan. In some embodiments, the β-glucan is β-1,3;1,6-glucan. In someembodiments, the β-1,3;1,6-glucan is derived from baker's yeast. Thenutritional composition may comprise whole glucan particle β-glucan,particulate β-glucan, PGG-glucan(poly-1,6-β-D-glucopyranosyl-1,3-β-D-glucopyranose) or any mixturethereof.

In some embodiments, the amount of β-glucan present in the compositionis at between about 0.010 and about 0.080 g per 100 g of composition. Inother embodiments, the nutritional composition comprises between about10 and about 30 mg β-glucan per serving. In another embodiment, thenutritional composition comprises between about 5 and about 30 mgβ-glucan per 8 fl. oz. (236.6 mL) serving. In other embodiments, thenutritional composition comprises an amount of β-glucan sufficient toprovide between about 15 mg and about 90 mg β-glucan per day. Thenutritional composition may be delivered in multiple doses to reach atarget amount of β-glucan delivered to the subject throughout the day.

In some embodiments, the amount of β-glucan in the nutritionalcomposition is between about 3 mg and about 17 mg per 100 kcal. Inanother embodiment the amount of β-glucan is between about 6 mg andabout 17 mg per 100 kcal.

The nutritional composition of this disclosure may also includephosphatidylethanolamine (“PE”), recognized as havingneurogenesis-promoting effects, particularly in infants, as taught bySer. No. 13/739,787, filed Jan. 11, 2013. It is believed that PE maysynergistically work with inositol to enhance the effects noted herein.Examples of PE suitable for inclusion in the neurologic componentinclude, but are not limited to,1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine,1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine,1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine,1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine,1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine,1,2-Distearoyl-sn-glycero-3-phosphoethanolamine,1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine,1-arachidonoyl-2-stearoyl-sn-glycerol 3-phospho ethanolamine,N,1-Diarachidonoyl-2-stearoyl-sn-glycerol 3-phosphoethanolamine, andphosphoethanolanime containing any fatty acid at the 1 and/or 2positions.

PE may be present, in some embodiments, in an amount from about 3.7mg/100 kcal to about 37 mg/100 kcal. In other embodiments, PE may bepresent from about 10 mg/100 kcal to about 30 mg/100 kcal. In stillother embodiments, PE may be present from about 15 mg/100 kcal to about25 mg/100 kcal.

Sphingomyelin has also been recognized as having neurogenesis-promotingeffects, particularly in infants, as taught by Ser. No. 13/739,787,filed Jan. 11, 2013, and can be incorporated into the nutritionalcomposition of the present disclosure in certain embodiments, tosynergistically combine with inositol to further improve the disclosedneurological benefits. Sphingomyelin refers to a class of sphingolipidsfound in animal cell membranes, particularly in the myelin sheath thatsurrounds nervous cell axons. In humans, sphingomyelin typically makesup 10% to 20% of plasma membrane lipids. It is believed thatsphingomyelin serves to electrically insulate nerve cell axons as itmakes up 25% the total lipids in the myelin sheath that surround andinsulate cells of the central nervous system. Membrane sphingomyelin isthe precursor for sphingosine which is the precursor forsphingosine-1-phosphate, which may have a role in neurogenesis and isneuroprotective. Sphingosine-1-phosphate may also facilitate neuralstem/progenitor cell (“NSPC”) migration, which is essential to thedevelopment of the nervous system as well as the ongoing neurogenesisthat occurs in the mature central nervous system.

Examples of sphingomyelin suitable for inclusion in the neurologiccomponent of the nutritional composition include, but are not limited toceramide phosphorylcholine and ceramide phosphorylethanolamine, N-oleoylsphingomyelin, N-stearoyl sphingomyelin, and/or D-erythro N-palmitoylsphingomyelin, and mixtures thereof. For example, in one embodiment thesphingomyelin included in the neurologic component may be syntheticsphingomyelin prepared according to the procedures of U.S. Pat. No.7,687,652 to Rochlin et al., however, the present disclosure can alsoinclude other processes for production of synthetic sphingomyelin.

When present, sphingomyelin can be incorporated at a level of about 0.15mg/100 kcal to about 73 mg/100 kcal.

Alpha-lipoic acid (ALA) can also be incorporated into the nutritionalcomposition of the present disclosure in some embodiments. ALA has beenrecognized as having neurogenesis-promoting effects, particularly ininfants, as taught by Ser. No. 13/942,794, filed Jul. 16, 2013. ALA may,under certain circumstances, synergistically combine with inositol tofurther improve the neurological benefits of inositol. Examples of ALAsuitable for use herein include, but are not limited to, enantiomers andracemic mixtures of ALA, including, R-lipoic acid “RLA”, S-lipoic acid“SLA”, and R/S-LA. Also suitable is R-lipoic acid stabilized with eithersodium (“Na-RALA”) or potassium as Potassium-R-Lipoate.

When incorporated into the disclosed nutritional composition, ALA may bepresent, in some embodiments, in an amount from about 0.1 mg/100 kcal toabout 35 mg/100 kcal. In some embodiments, ALA may be present in anamount from about 2.0 mg/100 kcal to about 25 mg/100 kcal. In stillother embodiments, ALA may be present in an amount from about 5.0 mg/100kcal to about 15 mg/100 kcal.

In certain embodiments, the nutritional composition of the presentdisclosure further comprises epigallocatchin-gallate (EGCG), which hasbeen recognized as having neurogenesis-promoting effects, particularlyin infants, as taught by Ser. No. 14/044,913, filed Oct. 3, 2013. It isbelieved that EGCG may synergistically combine with inositol to furtherimprove the neurological benefits of inositol, especially when EGCG ispresent in the disclosed nutritional composition at a level of about 5mg/100 kcal to about 120 mg/100 kcal.

Sulforaphane, which includes L-sulforaphane, may be incorporated intothe nutritional composition in an amount from about 1.5 mg/100 kcal toabout 7.5 mg/100 kcal. Still in some embodiments, sulforaphane may bepresent in an amount from about 2 mg/100 kcal to about 6 mg/100 kcal. Insome embodiments, sulforaphane may be present in an amount from about 3mg/100 kcal to about 5 mg/100 kcal. Sulforaphane has been recognized ashaving neurogenesis-promoting effects, particularly in infants, astaught by Ser. No. 13/942,794, filed Jul. 16, 2013, and may also exhibitsynergy with inositol herein.

One or more vitamins and/or minerals may also be added in to thenutritional composition in amounts sufficient to supply the dailynutritional requirements of a subject. It is to be understood by one ofordinary skill in the art that vitamin and mineral requirements willvary, for example, based on the age of the child. For instance, aninfant may have different vitamin and mineral requirements than a childbetween the ages of one and thirteen years. Thus, the embodiments arenot intended to limit the nutritional composition to a particular agegroup but, rather, to provide a range of acceptable vitamin and mineralcomponents.

The nutritional composition may optionally include, but is not limitedto, one or more of the following vitamins or derivations thereof:vitamin B₁ (thiamin, thiamin pyrophosphate, TPP, thiamin triphosphate,TTP, thiamin hydrochloride, thiamin mononitrate), vitamin B₂(riboflavin, flavin mononucleotide, FMN, flavin adenine dinucleotide,FAD, lactoflavin, ovoflavin), vitamin B₃ (niacin, nicotinic acid,nicotinamide, niacinamide, nicotinamide adenine dinucleotide, NAD,nicotinic acid mononucleotide, NicMN, pyridine-3-carboxylic acid),vitamin B₃-precursor tryptophan, vitamin B₆ (pyridoxine, pyridoxal,pyridoxamine, pyridoxine hydrochloride), pantothenic acid (pantothenate,panthenol), folate (folic acid, folacin, pteroylglutamic acid), vitaminB₁₂ (cobalamin, methylcobalamin, deoxyadenosylcobalamin, cyanocobalamin,hydroxycobalamin, adenosylcobalamin), biotin, vitamin C (ascorbic acid),vitamin A (retinol, retinyl acetate, retinyl palmitate, retinyl esterswith other long-chain fatty acids, retinal, retinoic acid, retinolesters), vitamin D (calciferol, cholecalciferol, vitamin D₃,1,25,-dihydroxyvitamin D), vitamin E (α-tocopherol, α-tocopherolacetate, α-tocopherol succinate, α-tocopherol nicotinate, α-tocopherol),vitamin K (vitamin K₁, phylloquinone, naphthoquinone, vitamin K₂,menaquinone-7, vitamin K₃, menaquinone-4, menadione, menaquinone-8,menaquinone-8H, menaquinone-9, menaquinone-9H, menaquinone-10,menaquinone-11, menaquinone-12, menaquinone-13), choline, inositol,β-carotene and any combinations thereof.

Further, the nutritional composition may optionally include, but is notlimited to, one or more of the following minerals or derivationsthereof: boron, calcium, calcium acetate, calcium gluconate, calciumchloride, calcium lactate, calcium phosphate, calcium sulfate, chloride,chromium, chromium chloride, chromium picolonate, copper, coppersulfate, copper gluconate, cupric sulfate, fluoride, iron, carbonyliron, ferric iron, ferrous fumarate, ferric orthophosphate, irontrituration, polysaccharide iron, iodide, iodine, magnesium, magnesiumcarbonate, magnesium hydroxide, magnesium oxide, magnesium stearate,magnesium sulfate, manganese, molybdenum, phosphorus, potassium,potassium phosphate, potassium iodide, potassium chloride, potassiumacetate, selenium, sulfur, sodium, docusate sodium, sodium chloride,sodium selenate, sodium molybdate, zinc, zinc oxide, zinc sulfate andmixtures thereof. Non-limiting exemplary derivatives of mineralcompounds include salts, alkaline salts, esters and chelates of anymineral compound.

The minerals can be added to nutritional compositions in the form ofsalts such as calcium phosphate, calcium glycerol phosphate, sodiumcitrate, potassium chloride, potassium phosphate, magnesium phosphate,ferrous sulfate, zinc sulfate, cupric sulfate, manganese sulfate, andsodium selenite. Additional vitamins and minerals can be added as knownwithin the art.

In an embodiment, the nutritional composition may contain between about10 and about 50% of the maximum dietary recommendation for any givencountry, or between about 10 and about 50% of the average dietaryrecommendation for a group of countries, per serving of vitamins A, C,and E, zinc, iron, iodine, selenium, and choline. In another embodiment,the children's nutritional composition may supply about 10-30% of themaximum dietary recommendation for any given country, or about 10-30% ofthe average dietary recommendation for a group of countries, per servingof B-vitamins. In yet another embodiment, the levels of vitamin D,calcium, magnesium, phosphorus, and potassium in the children'snutritional product may correspond with the average levels found inmilk. In other embodiments, other nutrients in the children'snutritional composition may be present at about 20% of the maximumdietary recommendation for any given country, or about 20% of theaverage dietary recommendation for a group of countries, per serving.

The nutritional compositions of the present disclosure may optionallyinclude one or more of the following flavoring agents, including, butnot limited to, flavored extracts, volatile oils, cocoa or chocolateflavorings, peanut butter flavoring, cookie crumbs, vanilla or anycommercially available flavoring. Examples of useful flavorings include,but are not limited to, pure anise extract, imitation banana extract,imitation cherry extract, chocolate extract, pure lemon extract, pureorange extract, pure peppermint extract, honey, imitation pineappleextract, imitation rum extract, imitation strawberry extract, or vanillaextract; or volatile oils, such as balm oil, bay oil, bergamot oil,cedarwood oil, cherry oil, cinnamon oil, clove oil, or peppermint oil;peanut butter, chocolate flavoring, vanilla cookie crumb, butterscotch,toffee, and mixtures thereof. The amounts of flavoring agent can varygreatly depending upon the flavoring agent used. The type and amount offlavoring agent can be selected as is known in the art.

The nutritional compositions of the present disclosure may optionallyinclude one or more emulsifiers that may be added for stability of thefinal product. Examples of suitable emulsifiers include, but are notlimited to, lecithin (e.g., from egg or soy), alpha lactalbumin and/ormono- and di-glycerides, and mixtures thereof. Other emulsifiers arereadily apparent to the skilled artisan and selection of suitableemulsifier(s) will depend, in part, upon the formulation and finalproduct.

The nutritional compositions of the present disclosure may optionallyinclude one or more preservatives that may also be added to extendproduct shelf life. Suitable preservatives include, but are not limitedto, potassium sorbate, sodium sorbate, potassium benzoate, sodiumbenzoate, calcium disodium EDTA, and mixtures thereof.

The nutritional compositions of the present disclosure may optionallyinclude one or more stabilizers. Suitable stabilizers for use inpracticing the nutritional composition of the present disclosureinclude, but are not limited to, gum arabic, gum ghatti, gum karaya, gumtragacanth, agar, furcellaran, guar gum, gellan gum, locust bean gum,pectin, low methoxyl pectin, gelatin, microcrystalline cellulose, CMC(sodium carboxymethylcellulose), methylcellulose hydroxypropyl methylcellulose, hydroxypropyl cellulose, DATEM (diacetyl tartaric acid estersof mono- and diglycerides), dextran, carrageenans, and mixtures thereof.

The disclosed nutritional composition(s) may be provided in any formknown in the art, such as a powder, a gel, a suspension, a paste, asolid, a liquid, a liquid concentrate, a reconstituteable powdered milksubstitute or a ready-to-use product. The nutritional composition may,in certain embodiments, comprise a nutritional supplement, children'snutritional product, infant formula, human milk fortifier, growing-upmilk or any other nutritional composition designed for an infant or apediatric subject. Nutritional compositions of the present disclosureinclude, for example, orally-ingestible, health-promoting substancesincluding, for example, foods, beverages, tablets, capsules and powders.Moreover, the nutritional composition of the present disclosure may bestandardized to a specific caloric content, it may be provided as aready-to-use product, or it may be provided in a concentrated form. Insome embodiments, the nutritional composition is in powder form with aparticle size in the range of 5 μm to 1500 μm, more preferably in therange of 10 μm to 300 μm.

If the nutritional composition is in the form of a ready-to-use product,the osmolality of the nutritional composition may be between about 100and about 1100 mOsm/kg water, more typically about 200 to about 700mOsm/kg water.

The nutritional compositions of the disclosure may provide minimal,partial or total nutritional support. The compositions may benutritional supplements or meal replacements. The compositions may, butneed not, be nutritionally complete. In an embodiment, the nutritionalcomposition of the disclosure is nutritionally complete and containssuitable types and amounts of lipid, carbohydrate, protein, vitamins andminerals. The amount of lipid or fat typically can vary from about 1 toabout 7 g/100 kcal. The amount of protein typically can vary from about1 to about 7 g/100 kcal. The amount of carbohydrate typically can varyfrom about 6 to about 22 g/100 kcal.

The nutritional composition of the present disclosure may furtherinclude at least one additional phytonutrient, that is, anotherphytonutrient component in addition to the pectin and/or starchcomponents described hereinabove. Phytonutrients, or their derivatives,conjugated forms or precursors, that are identified in human milk arepreferred for inclusion in the nutritional composition. Typically,dietary sources of carotenoids and polyphenols are absorbed by a nursingmother and retained in milk, making them available to nursing infants.Addition of these phytonutrients to infant or children's formulas allowssuch formulas to mirror the composition and functionality of human milkand to promote general health and well being.

For example, in some embodiments, the nutritional composition of thepresent disclosure may comprise, in an 8 fl. oz. (236.6 mL) serving,between about 80 and about 300 mg anthocyanins, between about 100 andabout 600 mg proanthocyanidins, between about 50 and about 500 mgflavan-3-ols, or any combination or mixture thereof. In otherembodiments, the nutritional composition comprises apple extract, grapeseed extract, or a combination or mixture thereof. Further, the at leastone phytonutrient of the nutritional composition may be derived from anysingle or blend of fruit, grape seed and/or apple or tea extract(s).

For the purposes of this disclosure, additional phytonutrients may beadded to a nutritional composition in native, purified, encapsulatedand/or chemically or enzymatically-modified form so as to deliver thedesired sensory and stability properties. In the case of encapsulation,it is desirable that the encapsulated phytonutrients resist dissolutionwith water but are released upon reaching the small intestine. Thiscould be achieved by the application of enteric coatings, such ascross-linked alginate and others.

Examples of additional phytonutrients suitable for the nutritionalcomposition include, but are not limited to, anthocyanins,proanthocyanidins, flavan-3-ols (i.e. catechins, epicatechins, etc.),flavanones, flavonoids, isoflavonoids, stilbenoids (i.e. resveratrol,etc.)proanthocyanidins, anthocyanins, resveratrol, quercetin, curcumin,and/or any mixture thereof, as well as any possible combination ofphytonutrients in a purified or natural form. Certain components,especially plant-based components of the nutritional compositions mayprovide a source of phytonutrients.

Some amounts of phytonutrients may be inherently present in knowningredients, such as natural oils, that are commonly used to makenutritional compositions for pediatric subjects. These inherentphytonutrient(s) may be but are not necessarily considered part of thephytonutrient component described in the present disclosure. In someembodiments, the phytonutrient concentrations and ratios as describedherein are calculated based upon added and inherent phytonutrientsources. In other embodiments, the phytonutrient concentrations andratios as described herein are calculated based only upon addedphytonutrient sources.

In some embodiments, the nutritional composition comprises anthocyanins,such as, for example, glucosides of aurantinidin, cyanidin, delphinidin,europinidin, luteolinidin, pelargonidin, malvidin, peonidin, petunidin,and rosinidin. These and other anthocyanins suitable for use in thenutritional composition are found in a variety of plant sources.Anthocyanins may be derived from a single plant source or a combinationof plant sources. Non-limiting examples of plants rich in anthocyaninssuitable for use in the inventive composition include: berries (acai,grape, bilberry, blueberry, lingonberry, black currant, chokeberry,blackberry, raspberry, cherry, red currant, cranberry, crowberry,cloudberry, whortleberry, rowanberry), purple corn, purple potato,purple carrot, red sweet potato, red cabbage, eggplant.

In some embodiments, the nutritional composition of the presentdisclosure comprises proanthocyanidins, which include but are notlimited to flavan-3-ols and polymers of flavan-3-ols (e.g., catechins,epicatechins) with degrees of polymerization in the range of 2 to 11.Such compounds may be derived from a single plant source or acombination of plant sources. Non-limiting examples of plant sourcesrich in proanthocyanidins suitable for use in the disclosed nutritionalcomposition include: grape, grape skin, grape seed, green tea, blacktea, apple, pine bark, cinnamon, cocoa, bilberry, cranberry, blackcurrant chokeberry.

Non-limiting examples of flavan-3-ols which are suitable for use in thedisclosed nutritional composition include catechin, epicatechin,gallocatechin, epigallocatechin, epicatechin gallate,epicatechin-3-gallate, epigallocatechin and gallate. Plants rich in thesuitable flavan-3-ols include, but are not limited to, teas, red grapes,cocoa, green tea, apricot and apple.

Certain polyphenol compounds, in particular flavan-3-ols, may improvelearning and memory in a human subject by increasing brain blood flow,which is associated with an increase and sustained brain energy/nutrientdelivery as well as formation of new neurons. Polyphenols may alsoprovide neuroprotective actions and may increase both brainsynaptogenesis and antioxidant capability, thereby supporting optimalbrain development in younger children.

Preferred sources of flavan-3-ols for the nutritional compositioninclude at least one apple extract, at least one grape seed extract or amixture thereof. For apple extracts, flavan-3-ols are broken down intomonomers occurring in the range 4% to 20% and polymers in the range 80%to 96%. For grape seed extracts flavan-3-ols are broken down intomonomers (about 46%) and polymers (about 54%) of the total favan-3-olsand total polyphenolic content. Preferred degree of polymerization ofpolymeric flavan-3-ols is in the range of between about 2 and 11.Furthermore, apple and grape seed extracts may contain catechin,epicatechin, epigallocatechin, epicatechin gallate, epigallocatechingallate, polymeric proanthocyanidins, stilbenoids (i.e. resveratrol),flavonols (i.e. quercetin, myricetin), or any mixture thereof. Plantsources rich in flavan-3-ols include, but are not limited to apple,grape seed, grape, grape skin, tea (green or black), pine bark,cinnamon, cocoa, bilberry, cranberry, black currant, chokeberry.

If the nutritional composition is administered to a pediatric subject,an amount of flavan-3-ols, including monomeric flavan-3-ols, polymericflavan-3-ols or a combination thereof, ranging from between about 0.01mg and about 450 mg per day may be administered. In some cases, theamount of flavan-3-ols administered to an infant or child may range fromabout 0.01 mg to about 170 mg per day, from about 50 to about 450 mg perday, or from about 100 mg to about 300 mg per day.

In an embodiment of the disclosure, flavan-3-ols are present in thenutritional composition in an amount ranging from about 0.4 to about 3.8mg/g nutritional composition (about 9 to about 90 mg/100 kcal). Inanother embodiment, flavan-3-ols are present in an amount ranging fromabout 0.8 to about 2.5 mg/g nutritional composition (about 20 to about60 mg/100 kcal).

In some embodiments, the nutritional composition of the presentdisclosure comprises flavanones. Non-limiting examples of suitableflavanones include butin, eriodictyol, hesperetin, hesperidin,homeriodictyol, isosakuranetin, naringenin, naringin, pinocembrin,poncirin, sakuranetin, sakuranin, steurbin. Plant sources rich inflavanones include, but are not limited to orange, tangerine,grapefruit, lemon, lime. The nutritional composition may be formulatedto deliver between about 0.01 and about 150 mg flavanones per day.

Moreover, the nutritional composition may also comprise flavonols.Flavonols from plant or algae extracts may be used. Flavonols, such asishrhametin, kaempferol, myricetin, quercetin, may be included in thenutritional composition in amounts sufficient to deliver between about0.01 and 150 mg per day to a subject.

The phytonutrient component of the nutritional composition may alsocomprise phytonutrients that have been identified in human milk,including but not limited to naringenin, hesperetin, anthocyanins,quercetin, kaempferol, epicatechin, epigallocatechin,epicatechin-gallate, epigallocatechin-gallate or any combinationthereof. In certain embodiments, the nutritional composition comprisesbetween about 50 and about 2000 nmol/L epicatechin, between about 40 andabout 2000 nmol/L epicatechin gallate, between about 100 and about 4000nmol/L epigallocatechin gallate, between about 50 and about 2000 nmol/Lnaringenin, between about 5 and about 500 nmol/L kaempferol, betweenabout 40 and about 4000 nmol/L hesperetin, between about 25 and about2000 nmol/L anthocyanins, between about 25 and about 500 nmol/Lquercetin, or a mixture thereof. Furthermore, the nutritionalcomposition may comprise the metabolite(s) of a phytonutrient or of itsparent compound, or it may comprise other classes of dietaryphytonutrients, such as glucosinolate or sulforaphane.

In certain embodiments, the nutritional composition comprisescarotenoids, such as lutein, zeaxanthin, astaxanthin, lycopene,beta-carotene, alpha-carotene, gamma-carotene, and/orbeta-cryptoxanthin. Plant sources rich in carotenoids include, but arenot limited to kiwi, grapes, citrus, tomatoes, watermelons, papayas andother red fruits, or dark greens, such as kale, spinach, turnip greens,collard greens, romaine lettuce, broccoli, zucchini, garden peas andBrussels sprouts, spinach, carrots.

Humans cannot synthesize carotenoids, but over 34 carotenoids have beenidentified in human breast milk, including isomers and metabolites ofcertain carotenoids. In addition to their presence in breast milk,dietary carotenoids, such as alpha and beta-carotene, lycopene, lutein,zeaxanthin, astaxanthin, and cryptoxanthin are present in serum oflactating women and breastfed infants. Carotenoids in general have beenreported to improve cell-to-cell communication, promote immune function,support healthy respiratory health, protect skin from UV light damage,and have been linked to reduced risk of certain types of cancer, andall-cause mortality. Furthermore, dietary sources of carotenoids and/orpolyphenols are absorbed by human subjects, accumulated and retained inbreast milk, making them available to nursing infants. Thus, addition ofphytonutrients to infant formulas or children's products would bring theformulas closer in composition and functionality to human milk.

Flavonoids, as a whole, may also be included in the nutritionalcomposition, as flavonoids cannot be synthesized by humans. Moreover,flavonoids from plant or algae extracts may be useful in the monomer,dimer and/or polymer forms. In some embodiments, the nutritionalcomposition comprises levels of the monomeric forms of flavonoidssimilar to those in human milk during the first three months oflactation. Although flavonoid aglycones (monomers) have been identifiedin human milk samples, the conjugated forms of flavonoids and/or theirmetabolites may also be useful in the nutritional composition. Theflavonoids could be added in the following forms: free, glucuronides,methyl glucuronides, sulphates, and methyl sulphates.

The nutritional composition may also comprise isoflavonoids and/orisoflavones. Examples include, but are not limited to, genistein(genistin), daidzein (daidzin), glycitein, biochanin A, formononetin,coumestrol, irilone, orobol, pseudobaptigenin, anagyroidisoflavone A andB, calycosin, glycitein, irigenin, 5-O-methylgenistein, pratensein,prunetin, psi-tectorigenin, retusin, tectorigenin, iridin, ononin,puerarin, tectoridin, derrubone, luteone, wighteone, alpinumisoflavone,barbigerone, di-O-methylalpinumisoflavone, and4′-methyl-alpinumisoflavone. Plant sources rich in isoflavonoids,include, but are not limited to, soybeans, psoralea, kudzu, lupine,fava, chick pea, alfalfa, legumes and peanuts. The nutritionalcomposition may be formulated to deliver between about 0.01 and about150 mg isoflavones and/or isoflavonoids per day.

In an embodiment, the nutritional composition(s) of the presentdisclosure comprises an effective amount of choline. Choline is anutrient that is essential for normal function of cells. It is aprecursor for membrane phospholipids, and it accelerates the synthesisand release of acetylcholine, a neurotransmitter involved in memorystorage. Moreover, though not wishing to be bound by this or any othertheory, it is believed that dietary choline and docosahexaenoic acid(DHA) act synergistically to promote the biosynthesis ofphosphatidylcholine and thus help promote synaptogenesis in humansubjects. Additionally, choline and DHA may exhibit the synergisticeffect of promoting dendritic spine formation, which is important in themaintenance of established synaptic connections. In some embodiments,the nutritional composition(s) of the present disclosure includes aneffective amount of choline, which is about 20 mg choline per 8 fl. oz.(236.6 mL) serving to about 100 mg per 8 fl. oz. (236.6 mL) serving.

Moreover, in some embodiments, the nutritional composition isnutritionally complete, containing suitable types and amounts of lipids,carbohydrates, proteins, vitamins and minerals to be a subject's solesource of nutrition. Indeed, the nutritional composition may optionallyinclude any number of proteins, peptides, amino acids, fatty acids,probiotics and/or their metabolic by-products, prebiotics, carbohydratesand any other nutrient or other compound that may provide manynutritional and physiological benefits to a subject. Further, thenutritional composition of the present disclosure may comprise flavors,flavor enhancers, sweeteners, pigments, vitamins, minerals, therapeuticingredients, functional food ingredients, food ingredients, processingingredients or combinations thereof.

The present disclosure further provides a method for providingnutritional support to a subject. The method includes administering tothe subject an effective amount of the nutritional composition of thepresent disclosure.

The nutritional composition may be expelled directly into a subject'sintestinal tract. In some embodiments, the nutritional composition isexpelled directly into the gut. In some embodiments, the composition maybe formulated to be consumed or administered enterally under thesupervision of a physician and may be intended for the specific dietarymanagement of a disease or condition, such as celiac disease and/or foodallergy, for which distinctive nutritional requirements, based onrecognized scientific principles, are established by medical evaluation.

The nutritional composition of the present disclosure is not limited tocompositions comprising nutrients specifically listed herein. Anynutrients may be delivered as part of the composition for the purpose ofmeeting nutritional needs and/or in order to optimize the nutritionalstatus in a subject.

In some embodiments, the nutritional composition may be delivered to aninfant from birth until a time that matches full-term gestation. In someembodiments, the nutritional composition may be delivered to an infantuntil at least about three months corrected age. In another embodiment,the nutritional composition may be delivered to a subject as long as isnecessary to correct nutritional deficiencies. In yet anotherembodiment, the nutritional composition may be delivered to an infantfrom birth until at least about six months corrected age. In yet anotherembodiment, the nutritional composition may be delivered to an infantfrom birth until at least about one year corrected age.

The nutritional composition of the present disclosure may bestandardized to a specific caloric content, it may be provided as aready-to-use product, or it may be provided in a concentrated form.

In some embodiments, the nutritional composition of the presentdisclosure is a growing-up milk. Growing-up milks are fortifiedmilk-based beverages intended for children over 1 year of age (typicallyfrom 1-3 years of age, from 4-6 years of age or from 1-6 years of age).They are not medical foods and are not intended as a meal replacement ora supplement to address a particular nutritional deficiency. Instead,growing-up milks are designed with the intent to serve as a complementto a diverse diet to provide additional insurance that a child achievescontinual, daily intake of all essential vitamins and minerals,macronutrients plus additional functional dietary components, such asnon-essential nutrients that have purported health-promoting properties.

The exact composition of a nutritional composition according to thepresent disclosure can vary from market-to-market, depending on localregulations and dietary intake information of the population ofinterest. In some embodiments, nutritional compositions according to thedisclosure consist of a milk protein source, such as whole or skim milk,plus added sugar and sweeteners to achieve desired sensory properties,and added vitamins and minerals. The fat composition is typicallyderived from the milk raw materials. Total protein can be targeted tomatch that of human milk, cow milk or a lower value. Total carbohydrateis usually targeted to provide as little added sugar, such as sucrose orfructose, as possible to achieve an acceptable taste. Typically, VitaminA, calcium and Vitamin D are added at levels to match the nutrientcontribution of regional cow milk. Otherwise, in some embodiments,vitamins and minerals can be added at levels that provide approximately20% of the dietary reference intake (DRI) or 20% of the Daily Value (DV)per serving. Moreover, nutrient values can vary between marketsdepending on the identified nutritional needs of the intendedpopulation, raw material contributions and regional regulations.

In certain embodiments, the nutritional composition is hypoallergenic.In other embodiments, the nutritional composition is kosher. In stillfurther embodiments, the nutritional composition is a non-geneticallymodified product. In an embodiment, the nutritional formulation issucrose-free. The nutritional composition may also be lactose-free. Inother embodiments, the nutritional composition does not contain anymedium-chain triglyceride oil. In some embodiments, no carrageenan ispresent in the composition. In other embodiments, the nutritionalcomposition is free of all gums.

In some embodiments, the disclosure is directed to a staged nutritionalfeeding regimen for a pediatric subject, such as an infant or child,which includes a plurality of different nutritional compositionsaccording to the present disclosure. Each nutritional compositioncomprises a hydrolyzed protein, at least one pre-gelatinized starch, andat least one pectin. In certain embodiments, the nutritionalcompositions of the feeding regimen may also include a source of longchain polyunsaturated fatty acid, at least one prebiotic, an ironsource, a source of β-glucan, vitamins or minerals, lutein, zeaxanthin,or any other ingredient described hereinabove. The nutritionalcompositions described herein may be administered once per day or viaseveral administrations throughout the course of a day.

Examples are provided to illustrate some embodiments of the nutritionalcomposition of the present disclosure but should not be interpreted asany limitation thereon. Other embodiments within the scope of the claimsherein will be apparent to one skilled in the art from the considerationof the specification or practice of the nutritional composition ormethods disclosed herein. It is intended that the specification,together with the example, be considered to be exemplary only, with thescope and spirit of the disclosure being indicated by the claims whichfollow the example.

Example 1

FIG. 1 illustrates the functional implications of inositol inoligodendroglia survival and proliferation. Inositol was added topurified oligodendroglial cultures and cells were analyzed for effectson proliferation, survival and differentiation by immunohistochemistryunder the fluorescent microscopy. OPCs were analyzed by staining withantibody against PDGFRa and visualized in green fluorescence; andoligodendrocytes with that against MBP in red. As a result of thefindings, inositol supplementation in purified oligodendroglia culturessignificantly promotes oligodendroglial survival, resulting in aconsistent increase in the number of oligodendrocyte precursor cells andmature oligodendrocytes (FIGS. 2 and 3). Depletion of inositol in themedium results in a fifty percent loss of oligodendrocyte precursorcells and thirty percent loss in oligodendrocytes. More importantly, theOPC and oligodendrocyte health significantly increased when supplementedwith inositol at 200 μM compared to the 40 μM in the base medium.

Example 2

A culture condition containing 40 μM inositol in which the count of thenumbers of living OPCs was normalized as 1, as a baseline control. Thebeginnings of culture of OPCs are equal in terms of OPC number inculture dish in all conditions. Inositol was added to each OPC cultureat zero, 200 nM, 1 μM, 10 μM, 40 μM, 100 μM, and 200 μM. Since the OPCsonly respond to platelet-derived growth factor PDGF, a potent mitogen,for proliferation, the condition containing 40 μM inositol and PDGF wasset as a positive control. When compared to baseline control at 40 μMinositol, the quantification of OPCs were significantly decreased atzero, 200 nM, 1 μM and 10 μM (*p<0.05). Moreover, when increasinginositol to 200 μM, the OPCs number was significantly increased(*p<0.05). Such a positive effect reached about 70% of that in thepositive control. The overall response to inositol displayed in adose-dependent manner suggesting a true response to inositol. As aresult of the findings, it concluded that nutritional supplementation ofinositol significantly promotes oligodendroglial proliferation andsurvival. The results are graphically illustrated in FIG. 2

Example 3

As illustrated in FIG. 3, the 40 μM inositol level was set as a baselinecontrol normalized as “1”; with the addition of PDGF a positive controlfor comparison. In the absence and the lower presence of inositol 200nM, the quantification of Oligo numbers were significantly decreasedsignificantly when compared with the baseline at 40 μM inositol(*p<0.05). When increasing the amount of inositol, the Oligo numbersrise and appear a statistical significance at level of 200 μM (*p<0.05).It demonstrated a consistent increase in the number of oligodendrocyteprecursor cells and mature oligodendrocytes. Interestingly, the effectof inositol on Oligo number behaved differently from the conditioncontaining PDGF in which the Oligo number decreased significantly(**p<0.05). It may relate to the different mechanisms by which the OPCsrespond to higher level of inositol and to the PDGF. Taken together, itis a novel finding that inositol at higher level promotes OPCsdifferentiation into mature Oligo.

Example 4

FIG. 4 illustrates the utility of dual-fluorescence labeling of OPCs ingreen and oligodendrocytes (Oligo) in red (left), as well as myelindeposition along the DRG neuronal fibers in red (right). It is toexamine a dose-response of the inositol on oligodendrocyte-DRGcoculture. Inositol was added to purified oligodendrocyte-DRG coculturesand cells were analyzed for effects on proliferation, survivaldifferentiation and myelination. In the absence of inositol, althoughmyelin was deposited, the amount of red labeling and appearance ofmyelin were inappropriate, as myelin displaced as amassing, not properlywrapping around the fibers like an elongated red fluorescent stripe. Atthe baseline condition containing 40 μM inositol (middle panel), myelinwas wrapping around the neuronal fiber displaced as stripes in redfluorescence. In the high level of inositol at 200 μM, it wasdemonstrated that more myelin has been deposited along the neuronalfibers. Taken together, it demonstrates that inositol at higher levelsenhances myelin deposition.

Example 5

FIG. 5 illustrates the inositol effects on the control myelin extent ina dose-responsive manner. In the coculture system, neurofibers wereincluded to validate the effects on myelin extent. In the absence ofinositol, few myelin internodes were formed compared to the control basemedium at 40 μM. Additionally, oligodendrocytes formed more and longermyelin internodes at 200 μM compared to the controls. Taken together,oligodendroglia rely on neuronal inositol for survival, proliferation,differentiation and the instruction of the generation of the appropriatenumber of myeline internodes and the extent of wrapping.

Example 6

Primary hippocampal neurons were prepared from rats at E18. In brief,dissected hippocampi were incubated in 0.05% trypsin at 37° C. for 20minutes and plated at a density of ˜30,000 cells per coverslip.Dissociated cells were plated on poly-L-lysine and incubated in a cellculture incubator with 5.0% CO₂. Cytosine arabinoside was added at afinal concentration of 2 μM per well 2 days in vitro to prevent gliacell overgrowth. For studies of inositol, neurons were either grown ininositol-free medium or in inositol-free medium supplemented with 40 μminositol. After day 4 in vitro, neurons were grown either ininositol-free medium or in inositol-free medium supplemented with 40 μmor 200 μM inositol. At the first time of treatment at 4 days in vitro,the entire medium was exchanged for fresh medium containing nutrients,or control medium without nutrients. At the time of subsequent nutrientadditions, approximately ½ of the medium was exchanged and replaced withnutrient-containing medium or control medium without nutrients. Neuronswere processed for immunostaining at 14 days in vitro, i.e. after 10days of chronic treatment. The data show that Increasing inositol fromstandard 40 μM to 200 μM substantially elevates the density of synapticspecializations. We determined this both by automated quantitativeimmunostaining for the presynaptic marker Bassoon or excitatorypostsynaptic Homer, as shown in FIGS. 6 and 7 (p<0.05).

A strong inositol effect was also seen when the density of synapticsites where both Bassoon and Homer co-localize was measured, asillustrated in FIG. 8, suggesting the functional synaptic development(p<0.05). Further, the experiment supports that the size of postsynapticsites is larger when inositol is added, indicating more efficientsynaptic transmission (p<0.05) (see, FIG. 9).

Confocal imaging was performed on a Leica TCS SPE DM2500 microscopeequipped with one spectral PMT. Fluorochromes imaged include Alexa 488,Alexa 555, and Alexa 647 (Invitrogen). Green fluorescence representsBassoon presynaptic, red Homer postsynaptic, blue MAP2 dendrites.Depletion of inositol causes poor health of the neurons (right panel inFIG. 10). There was noticeable lack of growth and synapses which wasvisible while imaging. However, inositol significantly promotes theoverall health of neurons at 40 and 200 μM, respectively. Elevated levelof inositol supplement significantly enhances the formation offunctional synapses and overall neuron health (Left panel, in FIG. 10).

Example 7

This example illustrates an embodiment of a nutritional compositionaccording to the present disclosure.

Nutrient per 100 kcal Protein (g) 2 Fat (g) 5 Carbohydrates (g) 11Prebiotic (g) 0.6 Beta glucan (mg) 9 Polar lipids (mg) 100 Lactoferrin(mg) 90 Probiotic(s) (cfu) 1 × 10⁸ DHA (mg) 22 ARA (mg) 40 Vitamin A(IU) 400 Vitamin D (IU) 75 Vitamin E (IU) 2 Vitamin K (mcg) 12 Thiamin(mcg) 120 Riboflavin (mcg) 200 Vitamin B6 (mcg) 100 Vitamin B12 (mcg)0.5 Niacin (mcg) 1100 Folic acid (mcg) 20 Panthothenic acid (mcg) 600Biotin (mcg) 4 Vitamin C (mg) 18 Choline (mg) 30 Calcium (mg) 120Phosphorus (mg) 60 Sodium (mg) 28 Potassium (mg) 140 Chloride (mg) 100Iodine (mcg) 22 Iron (mg) 2 Zinc (mg) 1.2 Manganese (mcg) 25 Copper(mcg) 100 Selenium (mcg) 4 Endogenous Inositol (mg) 6 Exogenous Inositol(mg) 24 Carnitine (mg) 3 Taurine (mg) 8 Adenosine monophosphate (mg) 1Cytidine monophosphate (mg) 4 Guanosine monophosphate (mg) 0.8 Uridinemonophosphate (mg) 1

All references cited in this specification, including withoutlimitation, all papers, publications, patents, patent applications,presentations, texts, reports, manuscripts, brochures, books, internetpostings, journal articles, periodicals, and the like, are herebyincorporated by reference into this specification in their entireties.The discussion of the references herein is intended merely to summarizethe assertions made by their authors and no admission is made that anyreference constitutes prior art. Applicants reserve the right tochallenge the accuracy and pertinence of the cited references.

Although embodiments of the disclosure have been described usingspecific terms, devices, and methods, such description is forillustrative purposes only. The words used are words of descriptionrather than of limitation. It is to be understood that changes andvariations may be made by those of ordinary skill in the art withoutdeparting from the spirit or the scope of the present disclosure, whichis set forth in the following claims. In addition, it should beunderstood that aspects of the various embodiments may be interchangedin whole or in part. For example, while methods for the production of acommercially sterile liquid nutritional supplement made according tothose methods have been exemplified, other uses are contemplated.Therefore, the spirit and scope of the appended claims should not belimited to the description of the versions contained therein.

What is claimed is:
 1. A method for enhancing myelin deposition in apediatric subject, the method comprising administering to the pediatricsubject a nutritional composition comprising: about 2 g/100 kcal toabout 7 g/100 kcal of a fat or lipid; about 1 g/100 kcal to no greaterthan about 7 g/100 kcal of a protein or protein equivalent; about 5g/100 kcal to about 25 g/100 kcal of a carbohydrate; and about 12 mg/100kcal to about 40 mg/100 kcal of inositol, wherein the inositol comprisesexogenous inositol and inherent inositol, wherein a ratio of theexogenous inositol to the inherent inositol is at least 75:25, andwherein administration of the nutritional composition to the pediatricsubject enhances myelin deposition in the neurons of the pediatricsubject.
 2. The method of claim 1, wherein the nutritional compositionfurther comprises at least one long chain polyunsaturated fatty acid. 3.The method of claim 2, wherein the at least one long chainpolyunsaturated fatty acid includes at least one of docosahexaenoic acidor arachidonic acid.
 4. The method of claim 3, wherein the at least onelong chain polyunsaturated fatty acid is present from about 5 mg/100kcal to about 75 mg/100 kcal.
 5. The method of claim 1, wherein thenutritional composition further comprises docosahexaenoic acid,arachidonic acid, phosphatidylethanolamines, sphingomyelin, alpha-lipoicacid, epigallocatechin gallate, sulforaphane, or combinations thereof.6. The method of claim 1, wherein the nutritional composition furthercomprises lactoferrin present at a level of about 10 mg/100 kcal toabout 200 mg/100 kcal.
 7. The method of claim 1, wherein the nutritionalcomposition further comprises a prebiotic composition comprisingpolydextrose and galactooligosaccharides, wherein the prebioticcomponent comprises at least 20% w/w polydextrose andgalactooligosaccharides and mixtures thereof.
 8. The method of claim 1,wherein the nutritional composition is an infant formula or a growing upmilk.